A candidate molecule for the matrix assembly receptor to the N-terminal 29-kDa fragment of fibronectin in chick myoblasts.

564

567

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.

Section: Discussionmentioning

confidence: 99%

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

Zhong

Chrzanowska‐Wodnicka

Brown

et al. 1998

564

567

“…The "Fibronectin Assembly Receptor" Must be Subject to Rapid Up-and Down-regulation Studies of deletion and mutation sets of the recombinant fragment indicate that the amino-terminal five type I modules act as a unit that mediates binding to cells (Sottile et al, 1991). Proteins of 60-70 kD have been isolated from detergent extracts of macrophages (Blystone and Kaplan, 1992) and myoblasts (Moon et al, 1994) that bind to a subfragment of the 70-kD fragment that contains the five type I modules. Assembly of fibronectin requires fibronectin-fibronectin interactions, raising the possibility that the binding molecule on cell surfaces is fibronectin itself.…”

Section: The Effect Of Lpa On L~bronectin Binding Is Specific and Uniquementioning

confidence: 99%

Modulation of cell surface fibronectin assembly sites by lysophosphatidic acid.

Zhang

Checovich

Peters

et al. 1994

101

100

Abstract. Lysophosphatidic acid is a product of activated platelets and has diverse actions on cells. We have characterized the effect of lysophosphatidic acid on cell-mediated binding and assembly of fibronectin, an extracellular matrix protein. Serum made from whole blood, but neither platelet-poor plasma nor serum made from platelet-poor plasma, caused enhanced binding of fibronectin to cultured fibroblastic cells. The ability of whole blood serum to enhance binding of fibronectin was abolished by phospholipase B. These results indicate that lysophosphatidic acid derived from platelets is the principal component in whole blood serum that is active in the fibronectin binding assay. 1-oleoyl lysophosphatidic acid, 20-200 nM, was as active as 0.1-0.2% whole blood serum. The stimulatory effect of lysophosphatidic acid on the binding of fibronectin or the amino-terminal 70-kD fragment of fibronectin was rapid, sustained, and lost upon removal of lysophosphatidic acid. The stimulatory effect on binding could not be duplicated by bradykinin, platelet-activating factor, bombesin, or a peptide agonist of the thrombin receptor. Enhanced binding of the 70-kD fragment was due to increases in both the number and affinity of binding sites. Enhanced binding and assembly of fibronectin correlated with changes in cell shape and actin-containing cytoskeleton. The binding sites for fibronectin on lysophosphatidic acid-stimulated cells, as assessed by fluorescence, video, and scanning electron microscopy, were on areas of cell membrane containing numerous filopodia that extended between cells or between cells and substratum. These observations suggest that lysophosphatidic acid functions as a powerful and specific modulator of cell shape and early matrix assembly during wound healing.

“…The protein can polymerize into a disulfide-linked pericellular network in a multistep process which is under cellular control (Mosher et al, 1992). The nucleation step occurs on the cell surface and appears to involve several membrane components (Moon et al, 1994;Woods et al, 1988;Wu et al, 1993). Tumor cells do not assemble an FN network, a feature thought to be of importance for their invasive growth and formation of metastases (Giancotti and Ruoslahti, 1990;Hynes, 1990).…”

mentioning

confidence: 99%

Beta 1 integrin-dependent and -independent polymerization of fibronectin.

Wennerberg¹,

Lohikangas²,

Gullberg³

et al. 1996

273

297

Abstract. The mouse cell line GD25, which lacks expression of the 131 family of integrin heterodimers due to disruption of the 131 integrin subunit gene, was used for expression of full-length cDNA coding for splice variant A of the mouse 131 integrin subunit. In a stably transformed clone (GD25-131A), the expressed protein was found to form functional heterodimeric receptors together with the subunits et3, ~5, and et6. Both GD25 and GD25-131A attached to fibronectin and formed focal contacts which contained otv133, but no detectable ot5131A. The presence of GRGDS peptide allowed tx5131A to locate to focal contacts of GD25-131A cultured on fibronectin, while the 131-null GD25 cells were unable to attach under these conditions. Affinity chromatography revealed that ct5131A and otv133 could bind to a large cell-binding fragment of fibronectin, a5131A strongly promoted polymerization of fibronectin into a fibrillar network on top of the cells. Whereas little etv133 was colocalized with the fibronectin fibrils in GD25-131A cells, this integrin was able to support fibronectin fibril polymerization in GD25 cells. However, the etv133-induced polymerization was less efficient and occurred mainly in dense cultures of the GD25 cells. Thus, while both a5131A and ave3 are able to support adhesion to fibronectin, etv133 dominates in the formation of focal contacts, and a5131A has a prime function in fibronectin matrix assembly. This is the first report on fibronectin matrix assembly in the absence of 131 integrins.