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PubMed1984

Effect of acute plasma fibronectin depletion on tissue fibronectin levels: analysis by a new fluorescent immunoassay.

Plasma fibronectin modulates reticuloendothelial (RE) phagocytosis of cellular and tissue debris, fibrin microaggregates, and gelatin-coated particulates. An antigenically related, but more insoluble form of fibronectin is found in various tissues and suspected to play a role in vascular permeability, cell adhesion, and wound healing. The current study developed a fluorescent immunoassay which could be utilized for the quantification of tissue fibronectin following its extraction from tissues. Additionally, the changes in tissue fibronectin induced by the intravenous injection of gelatin-coated colloids in rats (300-350 g), which acutely depletes the plasma fibronectin level, were also investigated. Injection of gelatinized RE test lipid emulsion (50 mg/100 g) depleted the plasma fibronectin at 2 hr (80-85% depletion) followed by rebound restoration within 24 hr as quantified by either electroimmunoassay or fluorescent immunoassay. RES system clearance of the test particles from the blood resulted in an acute elevation in fibronectin extractable from the liver with a normalization by 48 hr. In contrast, assay of tissue fibronectin following a single extraction revealed a decrease in lung extractable fibronectin within 2 hr following RE blockade which persisted for 24-48 hr. Extractable fibronectin in spleen and renal tissue was unaltered by RE blockade. This microfluorescent immunoassay may provide a sensitive method to quantify fibronectin in small aliquots of tissue. Increased hepatic fibronectin most likely reflects interiorization of plasma fibronectin during Kupffer cell clearance of the test particles. Decreased lung extractable fibronectin may alter lung vascular sensitivity to a subsequent septic and/or intravascular coagulation stress. Thus, similar to the labile nature of plasma fibronectin, the concentration of fibronectin in various tissues, can undergo dynamic alterations.

Animals↗
PubMed1993

The alpha 5 beta 1 integrin fibronectin receptor, but not the alpha 5 cytoplasmic domain, functions in an early and essential step in fibronectin matrix assembly.

The alpha 5 beta 1 integrin mediates cell adhesion and migration on fibronectin, a glycoprotein critical for normal vertebrate embryonic development. Indirect evidence reported to date suggests that this receptor also functions in the deposition of fibronectin matrices. We used a molecular genetic approach to critically evaluate this role of alpha 5 beta 1 integrins. Mutant Chinese hamster ovary (CHO) cells deficient in alpha 5 integrin expression could not assemble a fibronectin matrix. Reconstituting alpha 5 beta 1 integrin expression by transfecting them with a full-length cDNA encoding the human alpha 5 chain completely restored fibronectin matrix assembly. CHO cells expressing an alpha 5 chain lacking the cytoplasmic domain also assembled a fibronectin matrix. Removing the cytoplasmic domain of alpha 5 appears to increase its activity in fibronectin matrix assembly. In addition to alpha 5 beta 1 integrin binding to fibronectin's RGD-containing domain, cells must bind with high affinity to fibronectin's amino-terminal 29-kDa matrix assembly domain to form a fibronectin matrix. Studies with the alpha 5-deficient CHO cells show that the expression of alpha 5 beta 1 integrin is also necessary for cells to bind fragments containing this distinct site in fibronectin and that a fibronectin matrix increases binding of the 29-kDa fragment. Thus, alpha 5 beta 1 integrins not only mediate cell adhesion to fibronectin, but also play an essential role in the assembly of a fibronectin matrix. This role includes direct binding to fibronectin and modulating a distinct binding event involving the interaction of fibronectin's amino-terminal matrix assembly domain with the cell surface.

Amino Acid Sequence↗
PubMed1989

Contribution of hepatic fibronectin synthesis to regulation of plasma fibronectin.

Intravenous injection of gelatinized particles, which are phagocytized by the reticuloendothelial system, elicits an acute depletion of plasma fibronectin followed by restoration to normal concentrations in 6-8 h and a rebound elevation at 24 h. We determined the contribution of hepatic fibronectin synthesis to the restoration of plasma fibronectin in rats after particle infusion by measuring the net incorporation of 75selenomethionine into plasma fibronectin during the recovery period. Rats injected intravenously with gelatinized particles had a greater (P less than 0.01) incorporation of labeled 75selenomethionine into fibronectin but less (P less than 0.05) incorporation of 75selenomethionine into total plasma protein than control rats. Inhibition (86%) of hepatic fibronectin synthesis by pretreatment with cycloheximide limited the recovery of fibronectin levels by only 60%, suggesting a source(s) other than hepatic synthesis may contribute to the restoration of plasma fibronectin. Using purified human plasma fibronectin as a tracer in the plasma pool, we found that 26% of the soluble fibronectin consumed from the plasma during particle clearance was subsequently released back into the plasma over a 4-h interval. Tissue analysis indicated that 125I-labeled fibronectin, which was previously incorporated into the tissues, was not released from the tissue pool after the injection of gelatinized particles. Thus the normalization and regulation of plasma fibronectin levels after its acute depletion due to blood-borne particles is a result of 1) an increase in hepatic synthesis of fibronectin, 2) the release of fibronectin previously consumed as an opsonin during particle clearance, and 3) the release of soluble intact fibronectin from a preformed storage pool.

Animals↗
PubMed1991

[Genesis of fibronectin in ascites--detection of cellular and plasma fibronectin in portal and malignant ascites].

Measurement of fibronectin in ascites has been proposed for the differentiation of ascites either due to malignant growth in the peritoneal cavity or liver cirrhosis with portal hypertension. The high ascitic fibronectin concentration in patients with peritoneal carcinomatosis was thought to be due to the synthesis of this protein by neoplastic cells. Therefore in ascites of malignant origin cellular fibronectin should be present as it is synthesized by neoplastic cells. On the other side the transsudative ascites due to liver cirrhosis with portal hypertension should mainly contain plasma-fibronectin, which is secreted by hepatocytes into the bloodstream. With the aid of two different monoclonal antibodies and immunoblotting of partially digested or intact ascitic fibronectin, cellular fibronectin could be demonstrated in ascitic fluid of 10 patients with peritoneal carcinomatosis, 13 patients with liver cirrhosis, one patient with right-sided heart failure and one patient with Budd-Chiari-Syndrome. As determined by a specific ELISA 8 out of 10 samples of malignant ascites contained more than 30 mg/l of cellular fibronectin, whereas 10 out of 13 samples of ascites due to liver cirrhosis contained less than 10 mg/l. Whereas in ascites of malignant origin cellular fibronectin represented about 20% of total fibronectin, in portal ascites fibronectin represented sometimes more than 50% of total fibronectin. Cellular fibronectin of non-malignant origin is probably produced by mesothelial cells or peritoneal macrophages. Therefore, fibronectin accumulating in peritoneal carcinomatosis is only to some extent locally produced, but mainly caused by an unhindered exsudation of plasma-fibronectin.

Adult↗
PubMed1991

Production of fibronectin by human tumor cells and interaction with exogenous fibronectin: comparison of cell lines obtained from colon adenocarcinomas and squamous carcinomas of the upper aerodigestive tract.

Cell lines derived from 13 different human colon adenocarcinomas were examined for production of fibronectin by ELISA and for cell-surface expression of fibronectin by indirect immunofluorescence. Two squamous epithelial cell lines obtained from tumors of the upper aerodigestive tract were used as controls. None of the 13 colon carcinoma lines produced detectable amounts of fibronectin or showed detectable cell-surface staining with anti-fibronectin. The 2 squamous epithelial cell lines, in contrast, produced large amounts of fibronectin which could be detected in the culture medium and bound to the substratum. The squamous carcinoma cells also stained brightly when examined in the viable state by immunofluorescence with anti-fibronectin. In addition to being studied for fibronectin production, each cell line was also examined for the ability to interact with exogenous fibronectin in an adhesion assay. None of the colon carcinoma cells were adherent to fibronectin-coated culture dishes while the 2 squamous carcinoma cells rapidly attached and spread on this substratum. These data suggest that cell lines derived from adenocarcinomas of the colon are deficient in production of fibronectin and in their ability to interact with exogenous fibronectin. In their degree of deficiency, the colon carcinoma cells are significantly different from several different types of human tumor cell. The failure of the colon carcinoma cells to synthesize detectable amounts of fibronectin endogenously or to interact with exogenous fibronectin may explain, in part, the low degree of adhesive interaction which these cells have for their substratum. This, in turn, may influence the in vitro and in vivo properties of colon carcinoma cells.

Adenocarcinoma↗
PubMed1996

Chick cartilage fibronectin differs in structure from the fibronectin in limb mesenchyme.

Fibronectin, present in the extracellular matrix of several tissues, is heterogeneous in structure. This heterogeneity is largely due to the alternative splicing of three exons (IIIB, IIIA, and V) during processing of the fibronectin primary transcript. Previously, we determined that the splicing patterns of fibronectin mRNAs change from B+A+ to B+A- during the differentiation of mesenchyme into cartilage (V. D. Bennett, K. M. Pallante, and S. L. Adams, J. Biol. Chem. 266, 5918-5924, 1991). Therefore, the structure of fibronectin at the protein level most likely changes during chondrogenesis as well. In order to characterize the fibronectin protein in chick limb prechondrogenic mesenchyme and cartilage, we generated a polyclonal antibody specific for the region encoded by exon IIIB in the chick fibronectin gene. Immunoblot and immunohistochemistry analyses with this antibody and an antibody specific for the region encoded by exon IIIA indicate that both antibodies react with the fibronectin present in prechondrogenic mesenchyme. In contrast, only the exon IIIB antibody reacts with the fibronectin present in chick cartilage. Quantitative ELISA assays with these antibodies indicate that approximately 96% of the fibronectin in chick embryonic cartilage contains exon IIIB while less than 3% of the fibronectin contains exon IIIA. These results corroborate the structures of the fibronectin isoforms present in prechondrogenic mesenchyme and cartilage that were predicted from our previous characterization of the fibronectin mRNAs in these tissues and indicate that essentially all of the fibronectin in cartilage is synthesized and secreted by cartilage chondrocytes.

Alternative Splicing↗
PubMed1988

Release of fibronectin fragments from endothelial cell monolayers exposed to activated leukocytes: relationship to plasma fibronectin levels after particle infusion.

Fibronectin is found in a soluble form in plasma as well as in an insoluble form in tissues. It is produced by cultured endothelial cells and can be localized in vitro and in vivo between adjacent endothelial cells as well as underneath endothelial cells in association with their collagenous matrix, where it is believed to influence cell adhesion. Fibronectin is susceptible to proteolytic cleavage by enzymes released from activated leukocytes. In the present study, cultured rat endothelial cells developed a fibrillar fibronectin network in their extracellular matrices in addition to releasing soluble, intact fibronectin (440 kDa) into their culture medium. Exposure of monolayers of cultured endothelial cells to activated polymorphonuclear leukocytes (PMNs) results in disruption of the fibrillar matrix fibronectin, damage to the endothelial cell monolayer, and presence of fibronectin fragments in the culture medium. In addition, acute leukocyte activation and peripheral leukopenia in vivo as induced by the intravenous infusion of foreign test particles also resulted in the appearance of low-molecular-weight fibronectin fragments in plasma. In the in vivo studies, the appearance of fibronectin fragments preceded the release of intact 440-kDa fibronectin in the plasma after its acute depletion by particle injection. Thus, activated leukocytes, adherent to an endothelial surface in vitro and in vivo, may result in degradation of matrix fibronectin and the release of fibronectin fragments into the extracellular environment. In vivo fibronectin fragments in blood may serve as a stimulus for the subsequent synthesis and/or release of intact plasma fibronectin.

Animals↗
PubMed1991

Incorporation of circulating fibronectin into various tissues during sepsis: colocalization with endogenous tissue fibronectin.

We studied the plasma clearance and tissue incorporation of intravenously infused purified human plasma fibronectin into various tissues during a period of acute lung vascular injury induced by lethal postoperative bacteremia in sheep. Lung, liver, spleen, and heart tissue were examined for both endogenous sheep tissue fibronectin as well as the experimentally infused human fibronectin using dual-label immunofluorescence. Awake sheep (n = 4) received a postoperative iv infusion of 5 x 10(9) live Pseudomonas over a 60-min infusion interval. Bacterial challenge was started 2 hr after starting the iv fibronectin infusion of purified human plasma fibronectin (100 mg iv bolus; 4 hr iv at 100 mg/hr). Human fibronectin displayed a biphasic rate of clearance from the plasma with entrance into lymph. Human fibronectin readily incorporated in all tissues studied, including the lung which was the focus of vascular injury. Analysis of tissue sections by dual-label immunofluorescence indicated that the exogenous human fibronectin colocalized with the endogenous sheep fibronectin. Thus, the plasma fibronectin concentration may influence the lung vascular barrier due to its incorporation into the tissue pool of fibronectin. Moreover, the plasma may serve as a reservoir for soluble fibronectin which can enter and colocalize with the insoluble tissue pool of fibronectin in various tissues.

Animals↗
PubMed2002

Interaction of fibrin(ogen) with fibronectin: further characterization and localization of the fibronectin-binding site.

The interaction of fibronectin with fibrin and its incorporation into fibrin clots are thought to be important for the formation of a provisional matrix that promotes cell adhesion and migration during wound healing. However, it is still unclear whether fibronectin interacts with both fibrin and fibrinogen or fibrin only and whether fibronectin binds exclusively to the fibrin(ogen) alphaC domains. To address these questions, we studied the interaction of fibronectin with fibrinogen, fibrin, and their proteolytic and recombinant fragments. In both ELISA and surface plasmon resonance (SPR) experiments, immobilized fibrinogen did not bind fibronectin at all, but after conversion to fibrin, it bound fibronectin with high affinity. To test which regions of fibrin are involved in this binding, we studied the interaction of fibronectin with the fibrin-derived D-D:E(1) complex and a recombinant alphaC fragment (residues Aalpha221-610) corresponding to the alphaC domain that together encompass the whole fibrin(ogen) molecule. In ELISA, when fibronectin was added to the immobilized D-D:E(1) complex or the immobilized alphaC fragment, only the latter exhibited binding. Likewise, when fibronectin was immobilized and the complex or the alphaC fragment was added, only the latter was observed to bind. The selective interaction between fibronectin and the alphaC fragment was confirmed by SPR. The fibronectin-binding site was further localized to the NH(2) terminal connector region of the alphaC domain since in ELISA, the immobilized recombinant Aalpha221-391 sub-fragment bound fibronectin well while the immobilized recombinant Aalpha392-610 sub-fragment exhibited no binding. This finding was confirmed by ligand blotting analysis. Thus, the results provide direct evidence for the existence of a cryptic high-affinity fibronectin-binding site in the Aalpha221-391 region of the fibrinogen alphaC domain that is not accessible in fibrinogen but becomes exposed in fibrin.

Binding Sites↗
PubMed1986

Regulation of fibronectin receptor distribution by transformation, exogenous fibronectin, and synthetic peptides.

Recent studies have shown that fibronectin and its 140K membrane receptor complex are spatially associated with microfilaments to form cell surface linkage complexes which are thought to mediate adhesive interactions between fibroblasts and their substrata. We examined the regulation of the organization of these cell surface structures in transformed and fibronectin-reconstituted cells as well as in cells treated with a competitive synthetic peptide inhibitor of fibronectin binding to its receptor. Correlative localization experiments with interference reflection microscopy and double-label or triple-label immunofluorescence revealed a concomitant loss of fibronectin, 140K receptor, and alpha-actinin colocalization at cell substratum extracellular matrix contact sites after transformation of chick fibroblasts by wild-type or temperature-sensitive Rous sarcoma viruses (RSV). Western and dot immunoblot analyses established that although similar total quantities of intact 140K molecules were present in the transformed cell cultures, significantly more was released into the culture medium of transformed cells. The 140K molecules on transformed cells were available for interaction with exogenously added fibronectin, which could reconstitute fibronectin-140K linkage complexes. In such fibronectin reconstitution experiments, many cells expressed both fibronectin-140K-actin linkage complexes and RSV pp60src, indicating that the morphological reversion could occur even in the continued presence of RSV transformation. The synthetic peptide Gly-Arg-Gly-Asp-Ser derived from the sequence of the cell-binding region of fibronectin could also prevent the organization of fibronectin-140K linkage complexes. Our results suggest that fibronectin interaction with cells regulates the organization of fibronectin receptor complexes and cytoskeletal components at the cell surface.

Actinin↗
PubMed1990

Fibronectin matrix deposition and fibronectin receptor expression in healing and normal skin.

During cutaneous tissue organization, numerous critical interactions occur between cells and the extracellular matrix (ECM). Cell-matrix interactions depend on the presence of ECM receptors. Many ECM receptors, known as integrins, are heterodimeric glycoproteins consisting of one alpha and one beta chain. Integrins containing beta 1 or beta 3 chains are ECM receptors, whereas those containing beta 2 chains are leukocyte cell-cell receptors. We have used porcine cutaneous wounds as a paradigm for tissue organization and probed healing wounds and adjacent normal skin with polyclonal antibodies to fibronectin and fibronectin (alpha 5 beta 1) receptor. During re-epithelialization, the epidermis transits over a provisional matrix containing fibronectin. Migrating epidermal cells expressed fibronectin receptors in a bright linear peripheral pattern. At 10 days, when reepithelialization was complete and the basement membrane was re-established, the fibronectin matrix was markedly reduced and fibronectin-receptor expression was limited to the basolateral aspect of basal cells, as observed in normal epidermis. Beneath the migrating epidermis in 5-d wounds, granulation tissue had filled 80% of the wound space. Day-5 wound fibroblasts did not express fibronectin nor other beta 1 integrin receptors, were randomly oriented, and contained no actin bundles. Fibronectin fibrils were assembled on the surfaces of day-5 wound fibroblasts but formed few linkages between cells. Day-7 wound fibroblasts expressed fibronectin receptors, contained peripheral cytoplasmic actin bundles consistent with a contractile fibroblast phenotype, and were coaligned across the wound in parallel array with interconnecting fibronectin fibrils. The wounds contracted between 7 and 10 days. Thus the migrating epidermis consistently expressed fibronectin receptors. Fibronectin receptors were expressed by fibroblasts just prior to wound contraction.

Animals↗
PubMed1994

Circulating fibronectin and fibronectin receptor in children with pertussis.

AIM: To determine concentrations of fibronectin and fibronectin receptor in children with pertussis. METHODS: Concentrations of circulating fibronectin and serum fibronectin receptor were detected in eight children affected by pertussis, eight children with acute upper or lower respiratory tract infections, and in 14 healthy control children. The single radial immunodiffusion technique and a solid phase enzyme immunoassay were used to detect circulating serum concentrations of fibronectin and fibronectin receptor. RESULTS: On admission, a significant decrease in fibronectin was detected in children with pertussis (p = 0.0006). Significant and decreased concentrations of fibronectin were also observed in children with upper or lower respiratory tract infections (p = 0.0002). On the other hand, serum fibronectin receptor concentrations were significantly increased in patients with pertussis, whereas patients with upper or lower respiratory tract infections had normal circulating fibronectin receptor concentrations. CONCLUSIONS: Fibronectin deficiency in children with pertussis may be related to diffusion and deposition of this protein in bronchial and alveolar spaces to limit infection, while increased fibronectin receptor concentrations are probably the expression of T cell activation and cell-mediated immunity during Bordetella pertussis infection.

Child, Preschool↗
PubMed1989

The localization of fibronectin and 140 Kd fibronectin receptor in the truncus arteriosus of the chick embryonic heart.

The distribution of fibronectin and 140 Kd fibronectin-receptor was examined in the truncus arteriosus of the chick embryonic heart during aortico-pulmonary (AP) septation by immunohistochemistry. In 4-day-old chick embryos, immunoreactivity of both anti-fibronectin and anti-140 Kd fibronectin-receptor was seen in the primordia of the AP septum. In 5-day-old embryos, cells stained with anti-140 Kd fibronectin-receptor antibody were rarely found in the AP septum, though strong immunoreactivity of the anti-fibronectin antibody was observed in the AP septum. In 6-day-old embryos, at a late stage of septation, cells stained with anti-140 Kd fibronectin-receptor antibody were not found in the AP septum, and immunoreactivity of the anti-fibronectin antibody had decreased. In the cushion tissue, immunoreactivity of the anti-fibronectin antibody was seen, but that of the anti-140 Kd fibronectin-receptor antibody was recognizable only on a few cushion cells in the 4-day-old embryos. These findings suggest that fibronectin is involved in the formation of the AP septum primordia, that fibronectin does not promote the development of the AP septum, and that the migration mechanism of cushion cells is different from that of neural crest cells.

Animals↗
PubMed1986

Human liver fibronectin complementary DNAs: identification of two different messenger RNAs possibly encoding the alpha and beta subunits of plasma fibronectin.

Human fibronectin polymorphism arises from variation in the C-terminal region [e.g., Sekiguchi, K., Siri, A., Zardi, L., & Hakomori, S. (1985) J. Biol. Chem. 260, 5105-5114]. In order to verify the chemical basis of the fibronectin polymorphism, cDNAs encoding the C-terminal region of human liver fibronectin have been isolated, sequenced, and compared with cDNAs encoding so-called "cellular fibronectin" (i.e., fibronectin produced by cultured cells in vitro). Among the five independent cDNAs thus isolated, two cDNAs, named pLF2 and pLF4, differed in the nucleotide sequence at the "type III connecting segment" (IIIcs) region. pLF4 contained 192 bases in this region whereas pLF2 completely lacked these bases. S1 mapping analysis indicated that both cDNAs with and without the 192 bases are faithful copies of two fibronectin mRNA species abundantly present in human liver. Comparison of the liver cDNAs with those coding for cellular fibronectin indicates that the latter cDNAs contain the 75-base and/or 93-base extra segments at the 5' and 3' boundaries of the 192-base IIIcs region. These extra segments have the consensus sequences for the 3' splice sites at their 3' ends, suggesting that fibronectin mRNAs with partial or complete deletion of the IIIcs sequence result from alternative splicing of a primary RNA transcript. Liver fibronectin cDNAs also lacked the 270-based "extra domain" (ED) segment present in some, but not all, cDNAs encoding cellular fibronectin. Thus, cellular fibronectin appears to have three extra peptide segments, encoded by the 75-base and 93-base segments in the IIIcs region and by the 270-base ED region, that are mostly absent in the liver fibronectin.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1983

Interaction of fibronectin with collagen: age-specific defect in the biological activity of human fibroblast fibronectin.

Fibronectins isolated fro early-passage and late-passage (in vitro aged) human fibroblasts were shown to differ in their ability to support cell adhesion and to influence cell morphology. Because fibroblast adhesion requires interactions between fibronectin, the cell surface, and the component of the extracellular matrix, we examined those functions in isolated cellular fibronectin. In comparison to fibronectin isolated from early-passage cells, fibronectin from late-passage cells bound poorly to native collagen types I and II. No differences were observed in the binding of the two fibronectins to denatured collagen. The binding of both fibronectins to native collagen was similarly promoted by heparin. Cell binding activity was evaluated by using a Boyden chamber assay to measure chemotaxis in response to either fibronectin. No differences were detected in cell binding. Comparisons of molecular weights by NaDodSO4/polyacrylamide gel electrophoresis reveals that fibronectin from late-passage cells is larger than that from early-passage cells. That difference is observed both in fibronectins isolated from conditioned media and in fibronectins isolated from the cell layer. These data support the hypothesis that late-passage cells produce a structurally and functionally distinct fibronectin. The defective binding to native collagen may account for some aspects of the aged phenotype.

Cell Adhesion↗
PubMed1999

Tenascin-C inhibits beta1 integrin-dependent T lymphocyte adhesion to fibronectin through the binding of its fnIII 1-5 repeats to fibronectin.

The extracellular matrix consists of different proteins interacting to form a meshwork-like structure. T lymphocyte adhesion to individual matrix proteins is mainly regulated at the adhesion receptor level, but it is conceivable that the composition of the matrix itself may affect T lymphocyte adhesion to individual proteins. We have addressed the latter point by studying the effect of the matrix protein tenascin-C (TN-C) on T lymphocyte adhesion to fibronectin. Here we report that TN-C inhibits adhesion of T lymphocytes and MOLT-4 lymphoma cells to fibronectin. We demonstrate that a TN-C fragment consisting of fibronectin type III repeats 1-5 (TNfnIII 1-5) but not TNfnIII A-D and TNfnIII 6-8 inhibited alpha5beta1 and alpha4beta1 integrin-mediated T lymphocyte and MOLT-4 adhesion to fibronectin. At concentrations that did not inhibit adhesion, TNfnIII 1-5 still prevented MOLT-4 cells from spreading on fibronectin. Preincubation and co-immobilization of TNfnIII 1-5 with fibronectin was more effective in inhibiting MOLT-4 adhesion to fibronectin than soluble TNfnIII 1-5 present during the adhesion test. Using an enzyme-linked immunosorbent assay we could demonstrate binding of TNfnIII 1-5 to fibronectin and fibronectin fragments. Taken together, these data demonstrate that the TNfnIII 1-5 domain is implicated in the inhibition of T lymphocyte adhesion to fibronectin caused by TN-C, and indicate that this effect involves the binding of TN-C repeats TNfnIII 1-5 to fibronectin.

Binding Sites↗
PubMed1994

Regulation of fibronectin and laminin receptor expression, fibronectin and laminin secretion in human colon cancer cells by transforming growth factor-beta 1.

Transforming growth factor (TGF)-beta 1 modulates the expression of extracellular matrix (ECM) glycoproteins, fibronectin and laminin and the adhesion of Moser colon cancer cells to these glycoproteins. Since adhesion can be altered through expression of cell-surface receptors, binding affinities of adhesion molecules for receptors, or both, we investigated the effect of TGF-beta 1 on the binding properties of fibronectin and laminin to their cell-surface receptors by saturation binding and Scatchard analyses using radiolabeled fibronectin and laminin. Fibronectin bound to its cell-surface receptor with high affinity (Kd = 1.25 x 10(-9) M), Moser cells had approximately 7.1 x 10(4) fibronectin-binding sites per cell. TGF-beta 1 treatment rapidly up-modulated the number of cell-surface fibronectin-binding sites by 1.9-fold. The binding affinity of fibronectin for the receptor, however, was not altered. Laminin was found to bind to a higher-affinity and a lower-affinity receptor. Moser cells expressed approximately 1.1 x 10(3) higher-affinity laminin-binding sites and approximately 3.1 x 10(4) lower-affinity-binding sites per cell. TGF-beta 1 rapidly increased the expression of the higher-affinity sites 3-fold and the lower-affinity sites 5-fold. The binding affinity of both the higher-affinity and lower-affinity laminin receptors increased 3-fold after 2 and 6 hr of TGF-beta 1 treatment respectively. Concurrent with receptor modulation, TGF-beta 1 induced the secretion of fibronectin and laminin from Moser cells. Northern hybridization analyses showed a concurrent stimulation of the expression of the mRNAs for ligands (fibronectin and laminin) and the mRNAs for the integrin species of the fibronectin and laminin receptors (alpha 5 and alpha 6 subunits). Thus the production of fibronectin and laminin and the expression of their receptors were tightly co-regulated by TGF-beta 1.

Colonic Neoplasms↗