scholarly journals A fibronectin self-assembly site involved in fibronectin matrix assembly: reconstruction in a synthetic peptide.

1992 ◽  
Vol 118 (2) ◽  
pp. 421-429 ◽  
Author(s):  
A Morla ◽  
E Ruoslahti
Keyword(s):  
Extracellular Matrix ◽  
Binding Site ◽  
Self Assembly ◽  
Synthetic Peptide ◽  
Active Form ◽  
Matrix Form ◽  
Matrix Assembly ◽  
Type Iii ◽  
Fibronectin Matrix ◽  
Fibronectin Binding

The active form of fibronectin is its extracellular matrix form, which allows for the attachment of cells and influences both the growth and migration of cells. The matrix form is assembled by cells; however, many cells are defective in this regard. Several regions within fibronectin have been shown to play a role in matrix assembly by cells. One such region has been localized into the first type III repeat of fibronectin (Chernousov, M. A., F. J. Fogerty, V. E. Koteliansky, and D. F. Mosher. J. Biol. Chem. 266:10851-10858). We have identified this site as a fibronectin-fibronectin binding site and reproduced it as a synthetic peptide. This site is contained in a 14-kD fragment that corresponds to portions of the first two type III repeats. The 14-kD fragment was found to bind to cell monolayers and to inhibit fibronectin matrix assembly. The 14-kD fragment only slightly reduced the binding of fibronectin to cell surfaces but it significantly inhibited the subsequent incorporation of fibronectin into the extracellular matrix. The 14-kD fragment also bound to purified fibronectin and inhibited fibronectin-fibronectin binding. A synthetic 31-amino acid peptide (P1) representing a segment of the 14-kD fragment retained the ability to inhibit fibronectin-fibronectin binding. Peptide P1 specifically bound fibronectin from plasma in affinity chromatography, whereas a column containing another peptide from the 14-kD fragment did not. These results define a fibronectin-fibronectin binding site that appears to promote matrix assembly by allowing the assembly of fibronectin molecules into nascent fibrils. The 14-kD fragment and the P1 peptide that contain this site inhibit matrix assembly by competing for the fibronectin-fibronectin binding.

scholarly journals A novel fibronectin binding site required for fibronectin fibril growth during matrix assembly

2001 ◽  
Vol 154 (5) ◽  
pp. 1081-1088 ◽  
Author(s):  
Jan L. Sechler ◽  
Hongwei Rao ◽  
Anne Marie Cumiskey ◽  
Irbert Vega-Colón ◽  
Michael S. Smith ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Binding Site ◽  
Large Deletion ◽  
Specific Function ◽  
Binding Assays ◽  
Cell Binding ◽  
Matrix Assembly ◽  
Type Iii ◽  
Fibronectin Binding ◽  
Cell Binding Domain

Fibronectin (FN) assembly into a fibrillar extracellular matrix is a stepwise process requiring participation from multiple FN domains. Fibril formation is regulated in part by segments within the first seven type III repeats (III1–7). To define the specific function(s) of this region, recombinant FNs (recFNs) containing an overlapping set of deletions were tested for the ability to assemble into fibrils. Surprisingly, recFN lacking type III repeat III1 (FNΔIII1), which contains a cryptic FN binding site and has been suggested to be essential for fibril assembly, formed a matrix identical in all respects to a native FN matrix. Similarly, displacement of the cell binding domain in repeats III9–10 to a position close to the NH2-terminal assembly domain, as well as a large deletion spanning repeats III4–7, had no effect on assembly. In contrast, two deletions that included repeat III2, ΔIII1–2 and ΔIII2–5, caused significant reductions in fibril elongation, although binding of FN to the cell surface and initiation of assembly still proceeded. Using individual repeats in binding assays, we show that III2 but not III1 contains an FN binding site. Thus, these results pinpoint repeat III2 as an important module for FN–FN interactions during fibril growth.

scholarly journals A novel role for the integrin-binding III-10 module in fibronectin matrix assembly.

1996 ◽  
Vol 133 (2) ◽  
pp. 431-444 ◽  
Author(s):  
D C Hocking ◽  
R K Smith ◽  
P J McKeown-Longo
Keyword(s):  
Binding Site ◽  
Wound Repair ◽  
Solid Phase ◽  
Focal Adhesions ◽  
Amino Terminus ◽  
Matrix Assembly ◽  
Amino Terminal ◽  
Fibronectin Matrix ◽  
Beta 1 Integrin ◽  
Rgd Site

Fibronectin matrix assembly is a cell-dependent process which is upregulated in tissues at various times during development and wound repair to support the functions of cell adhesion, migration, and differentiation. Previous studies have demonstrated that the alpha 5 beta 1 integrin and fibronectin's amino terminus and III-1 module are important in fibronectin polymerization. We have recently shown that fibronectin's III-1 module contains a conformationally sensitive binding site for fibronectin's amino terminus (Hocking, D.C., J. Sottile, and P.J. McKeown-Longo. 1994. J. Biol. Chem. 269: 19183-19191). The present study was undertaken to define the relationship between the alpha 5 beta 1 integrin and fibronectin polymerization. Solid phase binding assays using recombinant III-10 and III-1 modules of human plasma fibronectin indicated that the III-10 module contains a conformation-dependent binding site for the III-1 module of fibronectin. Unfolded III-10 could support the formation of a ternary complex containing both III-1 and the amino-terminal 70-kD fragment, suggesting that the III-1 module can support the simultaneous binding of III-10 and 70 kD. Both unfolded III-10 and unfolded III-1 could support fibronectin binding, but only III-10 could promote the formation of disulfide-bonded multimers of fibronectin in the absence of cells. III-10-dependent multimer formation was inhibited by both the anti-III-1 monoclonal antibody, 9D2, and amino-terminal fragments of fibronectin. A fragment of III-10, termed III-10/A, was able to block matrix assembly in fibroblast monolayers. Similar results were obtained using the III-10A/RGE fragment, in which the RGD site had been mutated to RGE, indicating that III-I0/A was blocking matrix assembly by a mechanism distinct from disruption of integrin binding. Texas red-conjugated recombinant III-1,2 localized to beta 1-containing sites of focal adhesions on cells plated on fibronectin or the III-9,10 modules of fibronectin. Monoclonal antibodies against the III-1 or the III-9,10 modules of fibronectin blocked binding of III-1,2 to cells without disrupting focal adhesions. These data suggest that a role of the alpha 5 beta 1 integrin in matrix assembly is to regulate a series of sequential self-interactions which result in the polymerization of fibronectin.

scholarly journals Induction of fibronectin matrix assembly in human fibrosarcoma cells by dexamethasone.

1987 ◽  
Vol 104 (3) ◽  
pp. 601-610 ◽  
Author(s):  
P J McKeown-Longo ◽  
C A Etzler
Keyword(s):  
Extracellular Matrix ◽  
Surface Protein ◽  
Order Rate Constant ◽  
Binding Activity ◽  
Matrix Assembly ◽  
Cultured Fibroblasts ◽  
Fibrosarcoma Cells ◽  
The Matrix ◽  
Fibronectin Binding ◽  
Human Fibrosarcoma Cells

Previous studies have suggested that the assembly of fibronectin into the extracellular matrix of cultured fibroblasts is mediated by specific matrix assembly receptors that recognize a binding site in the amino terminus of the fibronectin molecule (McKeown-Longo, P.J., and D.F. Mosher, 1985, J. Cell Biol., 100:364-374). In the presence of dexamethasone, human fibrosarcoma cells (HT-1080) acquired the ability to specifically bind exogenous plasma fibronectin and incorporate it into a detergent-insoluble extracellular matrix. Dexamethasone-induced fibronectin binding to HT-1080 cells was time dependent, dose dependent, and inhibited by cycloheximide. Saturation binding curves indicated that dexamethasone induced the appearance of 7.7 X 10(4) matrix assembly receptors per cell. The induced receptors exhibited a dissociation constant (KD) for soluble fibronectin of 5.0 X 10(-8) M. In parallel experiments, normal fibroblasts exhibited 4.1 X 10(5) receptors (KD = 5.3 X 10(-8) M) per cell. In the presence of cycloheximide, the induced fibronectin-binding activity on HT-1080 cells returned to uninduced levels within 12 h. In contrast, fibronectin-binding activity on normal fibroblasts was stable in the presence of cycloheximide for up to 54 h. The first-order rate constant (Kt = 2.07 X 10(-4) min-1) for the transfer of receptor-bound fibronectin to extracellular matrix was four- to fivefold less than that for normal fibroblasts (Kt = 1.32 X 10(-3) min-1). Lactoperoxidase-catalyzed iodination of HT-1080 monolayers indicated that a 48,000-mol-wt cell surface protein was enhanced with dexamethasone. The results from these experiments suggest that dexamethasone induces functional matrix assembly receptors on the surface of HT-1080 cells; however, the rate of incorporation of fibronectin into the matrix is much slower than that of normal fibroblasts.

Interleukin-1β stimulates early myogenesis of mouse C2C12 myoblasts: the impact on myogenic regulatory factors, extracellular matrix components, IGF binding proteins and protein kinases

2013 ◽  
Vol 16 (2) ◽  
pp. 255-264 ◽  
Author(s):  
K. Grabiec ◽  
J. Tokarska ◽  
M. Milewska ◽  
M. Błaszczyk ◽  
M. Gajewska ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Myogenic Differentiation ◽  
Active Form ◽  
Cyclin B1 ◽  
Interleukin 1Β ◽  
Integrin Β1 ◽  
Matrix Assembly ◽  
Igf Binding Proteins ◽  
Extracellular Matrix Components ◽  
The Impact

AbstractThe purpose of the study was to examine the mechanisms important for early myogenesis in mouse C2C12 myogenic cells exposed to interleukin-1β. Cyclin A and cyclin B1 were increased by interleukin-1β (1 ng/ml), but the level of cyclin D1 and total DNA content was unaffected. Fusion index and the rate of protein synthesis was increased in the presence of IL-1β, but these effects were limited to 3-day-treatment. IL-1β increased the level of MyoD, myogenin and MHC on the 3rd day of differentiation, without altering the content of the active form of myostatin, as well as it augmented the level of fibronectin, integrin β1 and full length 100 kDa form of ADAM12. IL-1β caused a decrease in IGFBP-4 and IGFBP-6 levels and a marked increase in IGFBP-5. The phosphorylation of PKB and ERK1/2 and the cellular content of p38 were elevated by IL-1β. We conclude that the myogenic effect of IL-1β was limited to the onset of myoblast fusion and was associated with: i) increase in the level of myogenic transcription factors i.e. MyoD and myogenin expression, ii) modification of extracellular matrix assembly and signaling, manifested by an increase in fibronectin, integrin-β1 and ADAM12 content, iii) drop in IGFBP-4 and IGFBP-6, and an increase in IGFBP-5, that could alter the local IGF-1 bioavailability, and iv) increase in phosphorylation of PKB and ERK1/2, and the expression of p38 kinase, leading to activation of intracellular pathways essential for myogenic differentiation.

The amino-terminal matrix assembly domain of fibronectin stabilizes cell shape and prevents cell cycle progression

1999 ◽  
Vol 112 (19) ◽  
pp. 3225-3235 ◽  
Author(s):  
R.A. Christopher ◽  
S.R. Judge ◽  
P.A. Vincent ◽  
P.J. Higgins ◽  
P.J. McKeown-Longo
Keyword(s):  
Cell Cycle ◽  
Extracellular Matrix ◽  
Cell Shape ◽  
Cellular Response ◽  
Cell Cycle Progression ◽  
Cytochalasin D ◽  
Matrix Assembly ◽  
Cycle Progression ◽  
Amino Terminal ◽  
Fibronectin Matrix

Adhesion to the extracellular matrix modulates the cellular response to growth factors and is critical for cell cycle progression. The present study was designed to address the relationship between fibronectin matrix assembly and cell shape or shape dependent cellular processes. The binding of fibronectin's amino-terminal matrix assembly domain to adherent cells represents the initial step in the assembly of exogenous fibronectin into the extracellular matrix. When added to monolayers of pulmonary artery endothelial cells, the 70 kDa fragment of fibronectin (which contains the matrix assembly domain) stabilized both the extracellular fibronectin matrix as well as the actin cytoskeleton against cytochalasin D-mediated structural reorganization. This activity appeared to require specific fibronectin sequences as fibronectin fragments containing the cell adhesion domain as well as purified vitronectin were ineffective inhibitors of cytochalasin D-induced cytoarchitectural restructuring. Such pronounced morphologic consequences associated with exposure to the 70 kDa fragment suggested that this region of the fibronectin molecule may affect specific growth traits known to be influenced by cell shape. To assess this possibility, the 70 kDa fragment was added to scrape-wounded monolayers of bovine microvessel endothelium and the effects on two shape-dependent processes (i.e. migration and proliferation) were measured as a function of time after injury and location from the wound. The addition of amino-terminal fragments of fibronectin to the monolayer significantly inhibited (by >50%) wound closure. Staining of wounded monolayers with BrdU, moreover, indicated that either the 70 kDa or 25 kDa amino-terminal fragments of fibronectin, but not the 40 kDa collagen binding fragment, also inhibited cell cycle progression. These results suggest that the binding of fibronectin's amino-terminal region to endothelial cell layers inhibits cell cycle progression by stabilizing cell shape.

Fibronectin matrix assembly enhances adhesion-dependent cell growth

1998 ◽  
Vol 111 (19) ◽  
pp. 2933-2943 ◽  
Author(s):  
J. Sottile ◽  
D.C. Hocking ◽  
P.J. Swiatek
Keyword(s):  
Extracellular Matrix ◽  
Cell Growth ◽  
Wound Repair ◽  
Focal Adhesions ◽  
Specific Cell ◽  
Embryonic Cells ◽  
Matrix Assembly ◽  
Cell Functions ◽  
Fibronectin Matrix ◽  
Adhesive Interactions

Cell growth control in non-transformed cells depends, in part, on adhesive interactions with the extracellular matrix. Following injury, excess or altered fibronectin deposition into the extracellular matrix may contribute to the pathogenesis of fibrosis and atherosclerosis by triggering changes in specific cell functions associated with wound repair, including cell proliferation and migration. To assess the role of fibronectin polymerization on cell growth, we isolated mouse embryonic cells that lack endogenous fibronectin (fibronectin-null cells) and established them in culture under serum-free conditions. These fibronectin-null cells do not produce any detectable fibronectin, but are capable of assembling a fibronectin matrix when cultured in the presence of exogenously added fibronectin. Our data indicate that adhesion-dependent growth in fibronectin-null cells is dramatically increased (>2-5x) by culturing cells in the presence of fibronectin. This fibronectin-induced cell growth was blocked by inhibiting fibronectin matrix assembly. Arg-Gly-Asp peptides or fragments of fibronectin that contain the Arg-Gly-Asp cell binding site promoted clustering of the (α)5beta1 integrin in focal adhesions, but did not enhance cell growth. These data indicate that the polymerization of fibronectin into the extracellular matrix positively regulates cell growth, and that occupancy and clustering of fibronectin-binding integrins alone are not sufficient to trigger increased cell growth.

scholarly journals Rho-mediated Contractility Exposes a Cryptic Site in Fibronectin and Induces Fibronectin Matrix Assembly

1998 ◽  
Vol 141 (2) ◽  
pp. 539-551 ◽  
Author(s):  
Cuiling Zhong ◽  
Magdalena Chrzanowska-Wodnicka ◽  
James Brown ◽  
Amy Shaub ◽  
Alexey M. Belkin ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Lysophosphatidic Acid ◽  
Self Assembly ◽  
The State ◽  
Integrin Activation ◽  
Matrix Assembly ◽  
Cell Contractility ◽  
Fibronectin Fragment ◽  
Fibronectin Matrix ◽  
Fibronectin Assembly

Many factors influence the assembly of fibronectin into an insoluble fibrillar extracellular matrix. Previous work demonstrated that one component in serum that promotes the assembly of fibronectin is lysophosphatidic acid (Zhang, Q., W.J. Checovich, D.M. Peters, R.M. Albrecht, and D.F. Mosher. 1994. J. Cell Biol. 127:1447–1459). Here we show that C3 transferase, an inhibitor of the low molecular weight GTP-binding protein Rho, blocks the binding of fibronectin and the 70-kD NH2-terminal fibronectin fragment to cells and blocks the assembly of fibronectin into matrix induced by serum or lysophosphatidic acid. Microinjection of recombinant, constitutively active Rho into quiescent Swiss 3T3 cells promotes fibronectin matrix assembly by the injected cells. Investigating the mechanism by which Rho promotes fibronectin polymerization, we have used C3 to determine whether integrin activation is involved. Under conditions where C3 decreases fibronectin assembly we have only detected small changes in the state of integrin activation. However, several inhibitors of cellular contractility, that differ in their mode of action, inhibit cell binding of fibronectin and the 70-kD NH2-terminal fibronectin fragment, decrease fibronectin incorporation into the deoxycholate insoluble matrix, and prevent fibronectin's assembly into fibrils on the cell surface. Because Rho stimulates contractility, these results suggest that Rho-mediated contractility promotes assembly of fibronectin into a fibrillar matrix. One mechanism by which contractility could enhance fibronectin assembly is by tension exposing cryptic self-assembly sites within fibronectin that is being stretched. Exploring this possibility, we have found a monoclonal antibody, L8, that stains fibronectin matrices differentially depending on the state of cell contractility. L8 was previously shown to inhibit fibronectin matrix assembly (Chernousov, M.A., A.I. Faerman, M.G. Frid, O.Y. Printseva, and V.E. Koteliansky. 1987. FEBS (Fed. Eur. Biochem. Soc.) Lett. 217:124–128). When it is used to stain normal cultures that are developing tension, it reveals a matrix indistinguishable from that revealed by polyclonal anti-fibronectin antibodies. However, the staining of fibronectin matrices by L8 is reduced relative to the polyclonal antibody when the contractility of cells is inhibited by C3. We have investigated the consequences of mechanically stretching fibronectin in the absence of cells. Applying a 30–35% stretch to immobilized fibronectin induced binding of soluble fibronectin, 70-kD fibronectin fragment, and L8 monoclonal antibody. Together, these results provide evidence that self-assembly sites within fibronectin are exposed by tension.

Localization of fibronectin matrix assembly sites on fibroblasts and endothelial cells

1997 ◽  
Vol 110 (5) ◽  
pp. 569-581 ◽  
Author(s):  
R.A. Christopher ◽  
A.P. Kowalczyk ◽  
P.J. McKeown-Longo
Keyword(s):  
Extracellular Matrix ◽  
Binding Sites ◽  
Focal Adhesions ◽  
Cytochalasin D ◽  
Adherent Cells ◽  
Matrix Assembly ◽  
Fibronectin Matrix ◽  
Nucleation Sites ◽  
Beta1 Integrin ◽  
Texas Red

Polymerization of soluble fibronectin into extracellular matrix fibers occurs through the interaction between the amino terminus of fibronectin contained within a 70 kDa fragment and ‘matrix assembly sites’ on the cell surface. The present studies were performed to localize the ‘matrix assembly sites’ (defined by 70 kDa binding sites) on newly adherent cells and on cells containing preformed fibronectin matrix. Matrix nucleation sites on newly spread cells were visualized using Texas Red conjugated 70 kDa fragment and were found to colocalize with vinculin and substrate fibronectin fibrils. Cells plated onto vitronectin coated coverslips did not exhibit any 70 kDa binding sites although these cells were well-spread with fully developed focal adhesions. Time course studies indicated that 70 kDa binding sites could be detected on newly adherent cells within 30–40 minutes following cell plating onto fibronectin coated coverslips, prior to the reorganization of substrate fibronectin into fibrils. Similarly, exogenous fibronectin conjugated with Texas Red was also colocalized with vinculin when added to newly adherent cells. The disruption of actin filaments with cytochalasin D both prevented the expression of 70 kDa binding sites and also resulted in the loss of established 70 kDa binding sites on newly spread cells. After 3 days in culture, cells organized an extensive fibronectin matrix and 70 kDa was colocalized with two distinct types of matrix fibronectin fibers: fine linear cell-associated fibers which co-stained with the beta1 integrin and coarse extracellular fibers which did not stain for the beta1 integrin. There was also a third type of fibronectin fiber which was organized into a meshwork structure. There was no localization of either beta1 or 70 kDa to these structures. Treatment of 3-day cells with cytochalasin D resulted in the disruption of cell-matrix fibers and cell-associated 70 kDa binding sites. In contrast, the coarse extracellular matrix fibers as well as the meshwork fibers were unaffected by cytochalasin. In the presence of cytochalasin D, 70 kDa bound to sites which colocalized with the coarse extracellular matrix fibers. These data suggest that de novo assembly of fibronectin matrix occurs at sites of focal adhesion and as fibronectin polymerization proceeds, matrix nucleation sites colocalize along cell associated fibronectin fibers. At later times 70 kDa is localized to a subset of more mature fibronectin-containing fibers. These results suggest that there are at least three morphologically distinct 70 kDa binding sites on adherent cells: one which colocalizes with beta1 to focal adhesions, a second which colocalizes with beta1 and fibronectin in matrix contacts, and a third which localizes to extracellular matrix fibers.

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