Engineering and expression of a recombinant fusion protein possessing fibroblast growth factor-2 and fibronectin fragment

There is a synergistic effect between fibronectin and fibroblast growth factor 2 (FGF-2) on osteoblast cell adhesion through an extracellular, signal-regulated kinase pathway. Here we describe the engineering of a fusion protein containing fibronectin fragment (FNIII9-10) connected to the COOH-terminus of FGF-2. Purified FGF2-FNIII9-10 fusion protein exhibited a significant increase of cell adhesion and proliferation of MG63 cells compared with FNIII9-10 alone (p < 0.05).     

Enhanced fibronectin-mediated cell adhesion of human osteoblast by fibroblast growth factor, FGF-2

Using specific recombinant human fibronectin peptide (hFNIII9-10) that contains the binding site for integrin, we found that the fibroblast growth factor, FGF-2, enhances fibronectin-mediated adhesion in human osteoblast-like MG63 cells. The mechanism of the synergistic adhesion was due to the activation of extracellular-regulated kinase (ERK)-type MAPK upon interaction of integrin to hFNIII9-10 and its downstream activation of signaling pathways.     

Design and expression of oligomeric fibronectin fusion protein: a strategy for enhancing cell adhesion activity

Cell adhesion to extracellular matrices, including fibronectin, results in clustering of integrins in focal adhesions. To promote the clustering of fibronectin and thus enhance its activity at the sites of focal adhesion formation, we have engineered a fusion protein containing recombinant fibronectin fragment (hFN) connected to the tetramerization helix domain of lac repressor for oligomeric assembly. Purified Lac-hFN fusion protein exhibited significant increase of cell adhesion and proliferation of GF cells compared with hFN alone (p < 0.05).     

Proteolytically stabilizing fibronectin without compromising cell and gelatin binding activity

Excessive proteolytic degradation of fibronectin (FN) has been implicated in impaired tissue repair in chronic wounds. We previously reported two strategies for stabilizing FN against proteolytic degradation; the first conjugated polyethylene glycol (PEG) through cysteine residues and the second conjugated PEG chains of varying molecular weight on lysine residues. PEGylation of FN via lysine residues resulted in increased resistance to proteolysis with increasing PEG size, but an overall decrease in biological activity, as characterized by cell and gelatin binding. Our latest method to stabilize FN against proteolysis masks functional regions in the protein during lysine PEGylation. FN is PEGylated while it is bound to gelatin Sepharose beads with 2, 5, and 10 kDa PEG precursors. This results in partially PEGylated FN that is more stable than native FN and whose proteolytic stability increases with PEG molecular weight. Unlike completely PEGylated FN, partially PEGylated FN has cell adhesion, gelatin binding, and matrix assembly responses that are comparable to native FN. This is new evidence of how PEGylation variables can be used to stabilize FN while retaining its activity. The conjugates developed herein can be used to dissect molecular mechanisms mediated by FN stability and functionality, and address the problem of FN degradation in chronic wounds. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:277–288, 2015     

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

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

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

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

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

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

Latent TGF-beta binding protein-3 (LTBP-3) requires binding to TGF-beta for secretion

Latent transforming growth factor-beta (TGF-beta) binding protein (LTBP)-1, which is easily secreted, has been shown to enhance the secretion of TGF-beta. Here we show that another member of the LTBP family, LTBP-3, is not secreted by several cell types, but secretion occurs after coexpression with TGF-beta. The secretion of LTBP-3 requires complexing of LTBP-3 with Cys33 of the TGF-beta propeptide.     

Fibronectin and heparin binding domains of latent TGF-beta binding protein (LTBP)-4 mediate matrix targeting and cell adhesion

Latent transforming growth factor (TGF)-β binding proteins are extracellular matrix (ECM) proteins involved in the regulation of TGF-β sequestration and activation. In this study, we have identified binding domains in LTBP-4, which mediate matrix targeting and cell adhesion. LTBP-4 was found to possess heparin binding activity, especially in its N-terminal region. The C-terminal domain of LTBP-4 supported fibroblast adhesion, a property reduced by soluble heparin. In addition, we found that LTBP-4 binds directly to fibronectin (FN), which was indispensable for the matrix assembly of LTBP-4. The FN binding sites were also located in the N-terminal region. Interestingly, heparin was able to reduce the binding of LTBP-4 to FN. In fibroblast cultures, LTBP-4 colocalized first with FN and subsequently with fibrillin-1, pointing to a role for FN in the early assembly of LTBP-4. In FN −/− fibroblasts, LTBP-mediated ECM targeting was disturbed, resulting in increased TGF-β activity. These results revealed new molecular interactions which are evidently important for the ECM targeting, but which also are evidence of novel functions for LTBP-4 as an adhesion molecule.     

Characterization of a fibronectin‐binding protein from Lactobacillus casei BL23

Aims: To characterize the functionality of the Lactobacillus casei BL23 fbpA gene encoding a putative fibronectin‐binding protein. Methods and Results: Adhesion tests showed that L. casei BL23 binds immobilized and soluble fibronectin in a protease‐sensitive manner. A mutant with inactivated fbpA showed a decrease in binding to immobilized fibronectin and a strong reduction in the surface hydrophobicity as reflected by microbial adhesion to solvents test. However, minor effects were seen on adhesion to the human Caco‐2 or HT‐29 cell lines. Purified 6X(His)FbpA bound to immobilized fibronectin in a dose‐dependent manner. Western blot experiments with FbpA‐specific antibodies showed that FbpA could be extracted from the cell surface by LiCl treatment and that protease digestion of the cells reduced the amount of extracted FbpA. Furthermore, surface exposition of FbpA was detected in other L. casei strains by LiCl extraction and whole‐cell ELISA. Conclusions: FbpA can be found at the L. casei BL23 surface and participates in cell attachment to immobilized fibronectin. We showed that FbpA is an important, but not the only, factor contributing to fibronectin binding in BL23 strain. Significance and Impact of the Study: This is the first report showing the involvement of FbpA in fibronectin binding in L. casei BL23 and represents a new contribution to the study of attachment factors in probiotic bacteria.     

Transforming growth factor-beta 1 binds to immobilized fibronectin

We have characterized the interaction of homodimeric porcine transforming growth factor-beta 1 (TGF-beta 1) with affinity-purified human plasma fibronectin. Using a solid-phase binding assay, we have demonstrated that TGF-beta 1 binds to fibronectin immobilized on Immunlon ITM microtiter plates. TGF-beta 1 binding increased with time, reaching a plateau after 4-6 h, and was dependent upon the concentration of both labeled TGF-beta 1 and immobilized fibronectin present. The binding of radiolabeled TGF-beta 1 to fibronectin was saturable and was reduced 75% in the presence of a 100-fold excess of unlabeled TGF-beta 1. TGF-beta 1 bound to fibronectin with an association rate constant (Ka) of 2.96 x 10(3) M-1 s-1 and did not readily dissociate under various conditions. The binding of TGF-beta 1 to fibronectin was insensitive to variations in ionic strength over a range of 0.1-1.0 M NaCl and was relatively insensitive to divalent cation concentration in the range of 0.1-10.0 mM as well. These data suggest that the binding of TGF-beta 1 to fibronectin may not be dependent upon the interaction of charged amino acids within these two molecules. However, the binding of TGF-beta 1 to fibronectin was strongly pH-dependent and binding decreased dramatically below pH 4.0 and above pH 10.0, suggesting that charged amino acids may influence TGF-beta 1/fibronectin interactions. The association of TGF-beta 1 with immobilized fibronectin or other extracellular matrix components and subsequent dissociation under acidic conditions or by an as-yet-unidentified mechanism may play a role in the distribution and/or activity of this potent growth regulator at sites of tissue injury and inflammation in vivo.     

The Membrane-associated form of the DNA repair protein Ku is involved in cell adhesion to fibronectin

The Ku heterodimer (Ku70/Ku80) plays a central role in DNA double-strand breaks recognition and repair. However, Ku is expressed also on the surface of different types of cells along with its intracellular pool within the nucleus and the cytoplasm. Participation of membrane-associated Ku in cell-cell interaction has been reported recently. Here, we describe a novel function of cell-surface Ku as an adhesion receptor for fibronectin (Fn). The role of Ku in cell adhesion was investigated by comparing the Ku80 deficient Chinese hamster ovary (CHO) cell line, xrs-6, with clones transfected stably with either the hamster or human Ku80 cDNA. Ku expression in transfectant cells resulted in a significant increased adhesion on Fn and type IV collagen as compared to control cells. The observed increase in cell adhesion relied on Ku cell-surface expression, since antibodies directed against Ku70 or Ku80 subunit inhibited adhesion on Fn of Ku80, but not control vector, transfected xrs-6 cells. In addition, both Ku70 and Ku80 present a structural relationship with integrin I (or A) domains and the A1 and A3 domains of von Willebrand factor, domains known to be involved in Fn binding. Both Ku70 and Ku80 exhibit a complete set of residues compatible in their position and chemical nature with the formation of a metal ion-dependent adhesion (MIDAS) site implicated in ligand binding and integrin activation. Taken together, these functional and structural approaches support a new role for Ku as an adhesion receptor for Fn.     

Raf kinase inhibitor protein positively regulates cell-substratum adhesion while negatively regulating cell-cell adhesion

Raf kinase inhibitor protein (RKIP) regulates a number of cellular processes, including cell migration. Exploring the role of RKIP in cell adhesion, we found that overexpression of RKIP in Madin-Darby canine kidney (MDCK) epithelial cells increases adhesion to the substratum, while decreasing adhesion of the cells to one another. The level of the adherens junction protein E-cadherin declines profoundly, and there is loss of normal localization of the tight junction protein ZO-1, while expression of the cell-substratum adhesion protein beta1 integrin dramatically increases. The cells also display increased adhesion and spreading on multiple substrata, including collagen, gelatin, fibronectin and laminin. In three-dimensional culture, RKIP overexpression leads to marked cell elongation and extension of long membrane protrusions into the surrounding matrix, and the cells do not form hollow cysts. RKIP-overexpressing cells generate considerably more contractile traction force than do control cells. In contrast, RNA interference-based silencing of RKIP expression results in decreased cell-substratum adhesion in both MDCK and MCF7 human breast adenocarcinoma cells. Treatment of MDCK and MCF7 cells with locostatin, a direct inhibitor of RKIP and cell migration, also reduces cell-substratum adhesion. Silencing of RKIP expression in MCF7 cells leads to a reduction in the rate of wound closure in a scratch-wound assay, although not as pronounced as that previously reported for RKIP-knockdown MDCK cells. These results suggest that RKIP has important roles in the regulation of cell adhesion, positively controlling cell-substratum adhesion while negatively controlling cell-cell adhesion, and underscore the complex functions of RKIP in cell physiology.     

The extracellular matrix (mesoglea) of hydrozoan jellyfish and its ability to support cell adhesion and spreading

The outer mesoglea (extracellular matrix; ECM) of hydrozoan jellyfish was found to contain a species-specific meshwork of striated fibers of different diameters. In the mesoglea, molecules were identified which exhibit several features of well known vertebrate ECM: a laminin-like molecule which appears cross-shaped on electronmicrographs, a fibronectin-like molecule (both detectable by their immunoreactivity at the exumbrella side) and a species-specific collagen consisting of 3 different -chains of which at least 2 can be decorated with con A. The -chains are linked by disulfide bridges. Acetic acid extraction of the mesoglea and subsequent salt precipitation yields fibrils which appear banded in the electron microscope and support species-specific adhesion and spreading of isolated tissue. These precipitated fibrils are mainly composed of the disulfide-linked collagen.     

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

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

Mesangial cell apoptosis induced by a fibronectin fragment

We previously showed that in passive Heymann nephritis (PHN) rats, a large quantity of fibronectin (FN) fragments containing the central cell-binding (CCB) domain and adjacent domains are generated in the kidney and excreted into urine (Nishizawa et al., Biol Pharm Bull 1998; 21: 429–433). To ascertain whether the FN fragments could affect the progression of PHN, we investigated the effect of a 150 K FN fragment containing the CCB and carboxyl-terminal heparin-binding (Hep 2) domains on cultured rat mesangial cells. When rat mesangial cells cultured on FN-coated plates were exposed to the 150 K FN fragment, some mesangial cells detached from the FN substrate and then underwent apoptosis as judged by nuclear and DNA fragmentations. The 150 K FN fragment competitively inhibited the mesangial cell adhesion to the FN substrate in a dose-dependent manner. Furthermore, gelatinzymography of the conditioned medium of mesangial cells showed that the 150 K FN fragment induced and/or poteintiated the extracellular matrix (ECM)-degrading proteinases including matrix metalloproteinases (MMPs) of mesangial cells. These results indicate that the 150 K FN fragment may elicit mesangial cell apoptosis by disrupting the mesangial cell adhesion through two distinct ways: the inhibition of mesangial cell adhesion and the ECM-degradation by the 150 K FN fragment-induced MMPs. Thus, FN fragments containing the CCB and adjacent domains generated in the kidneys of PHN rats may be involved in the evolution of the renal injury.     

Human keratinocytes that have not terminally differentiated synthesize laminin and fibronectin but deposit only fibronectin in the pericellular matrix

Fibronectin and laminin production by human keratinocytes cultured in serum-free, low-calcium medium without a fibroblast feeder layer were examined by several techniques. By indirect immunofluorescence, fibronectin but not laminin appeared as short radial fibrils between the cells and the substratum, and in the pericellular matrix. Synthesis of fibronectin and laminin by 7-day keratinocyte cultures was determined by 18 hr 35S-methionine metabolic labeling followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. Fibronectin accounted for 2.9% of total synthesized protein, 26.5% of fluid phase protein secretion, and 4.3% of deposited ECM protein. In contrast, only 0.1% of the total synthesized protein was laminin, little (6.3%) of this product was secreted, and none of this product was deposited in the ECM. Our results indicate that human keratinocytes under culture conditions that prevent terminal differentiation in vitro can synthesize, secrete, and deposit fibronectin in the extracellular matrix. Although these cells synthesize laminin, they secrete very little and deposit no detectable laminin in the matrix under these culture conditions. From these data we believe that fibronectin may play an important role in the interaction of epidermal cells with connective tissue matrix during wound healing or morphogenesis in in vivo situations in which the epidermis is not terminally differentiated.     

Latent transforming growth factor-beta 1 associates to fibroblast extracellular matrix via latent TGF-beta binding protein

The role of latent transforming growth factor-beta (TGF-beta) binding protein (LTBP) in the association of TGF-beta 1 to the extracellular matrix of cultured fibroblasts and HT-1080 fibrosarcoma cells was studied by immunochemical methods. The matrices were isolated from the cells, and the levels of LTBP and TGF-beta 1 were estimated by immunoblotting and immunoprecipitation. LTBP, TGF-beta 1, and its propeptide (latency-associated peptide, LAP) were found to associate to the extracellular matrix. Immunoblotting analysis indicated that treatment of the cells with plasmin resulted in a concomitant time and dose dependent release of both LTBP and TGF-beta 1 from the extracellular matrix to the supernatant. Comparison of molecular weights suggested that plasmin treatment resulted in the cleavage of LTBP from the high molecular weight fibroblast form to a form resembling the low molecular weight LTBP found in platelets. Pulse- chase and immunoprecipitation analysis indicated that both the free form of LTBP and LTBP complexed to latent TGF-beta were efficiently incorporated in the extracellular matrix, from where both complexes were slowly released to the culture medium. Addition of plasmin to the chase solution resulted, however, in a rapid release of LTBP from the matrix. Fibroblast derived LTBP was found to associate to the matrix of HT-1080 cells in a plasmin sensitive manner as shown by immunoprecipitation analysis. These results suggest that the latent form of TGF-beta 1 associates with the extracellular matrix via LTBP, and that the release of latent TGF-beta 1 from the matrix is a consequence of proteolytic cleavage(s) of LTBP.     

The structure of fibronectin and its role in cellular adhesion

Fibronectin is a large, adhesive glycoprotein which is found in a number of locations, most notably on cell surfaces, in extracellular matrixes, and in blood. Fibronectin has been detected in all vertebrates tested and in many invertebrates. Its presence in sponges is significant because this suggests that fibronectin may have appeared very early in evolution, possibly with the most primitive multicellular organisms. Cellular and plasma fibronectins have many striking similarities. However, the locations of the polypcptide chain differences between these two proteins indicate that plasma fibronectin cannot be derived from cellular fibronectin by means of simple post-translational proteolysis. Instead, these different types of fibronectin may be products of different genes or of differentially spliced messenger RNA molecules. Amniotic fluid fibronectin is possibly a third form of the protein. Cellular and plasma fibronectins are composed of at least six protcaseresistant domains which contain specific binding sites for actin, gelatin, heparin, Staphylococcus aureus, transglutarninase, fibrin, DNA, and a cell surface receptor. The relative locations of these domains have been mapped in the primary structure of fibronectin. The cell surface receptor for fibronectin has not been positively identified, but may be a glycoprotein, a glycolipid, or a complex of the two. Although cell-substratum adhesion is mediated by fibronectin, the locations of the areas of closest approach of the cell to the substratum (the adhesion plaques) and fibronectin are not coincident under conditions of active cell growth. Under conditions of cell growth arrest in low scrum concentrations, some fibronectin may become localized at the adhesion plaques. Models describing the domain structure of fibronectin and the molecular organization of the adhesion plaque area are presented.