Stimulation of extracellular matrix remodeling by the first type III repeat in fibronectin

Abstract

The fibronectin matrix contains cryptic sites which are thought to modulate cellular biological responses. One of these sites, located in fibronectin's first type III repeat (III1c), influences signaling pathways that are relevant to cytoskeletal organization and cell cycle progression. The purpose of this study was to identify possible mechanisms responsible for the effects of III1c on cell behavior. Recombinant peptides representing various type III repeats of fibronectin were compared for their effects on fibronectin matrix organization and activation of intracellular signaling pathways. III1c and III13 but not III11c or III10 bound to monolayers of human skin fibroblasts in a dose- and time-dependent manner and were localized to the extracellular matrix. Binding of III13, but not III1c, to matrix was sensitive to heparitinase, suggesting that the association of III1c with the matrix was not dependent on heparan sulfate proteoglycans. Quantitative and morphological assessment indicated that, in contrast to previously published reports, the binding of III1c to cell layers did not result in the loss or disruption of matrix fibronectin. Binding of III1c but not III13 to the extracellular matrix did result in the loss of a conformationally sensitive epitope present within the EDA type III module of cellular fibronectin. III1c-induced loss of the EDA epitope did not require the presence of cells, occurred within 1 hour and was associated with the activation of p38 mitogen-activated protein kinase (MAPK) followed by the formation of filopodia. Maximal phosphorylation of p38 MAPK occurred within 1 hour, whereas cytoskeletal changes did not appear until 12 hours later. These findings are consistent with a model in which the binding of III1c to the extracellular matrix results in a conformational remodeling of the fibronectin matrix, which has both short- and long-term effects on cell physiology.

Domain Unfolding Plays a Role in Superfibronectin Formation

“…Our analysis identifies the segment III (20). This epitope loss was originally interpreted as remodeling of the matrix.…”

“…Although a high concentration of imidazole was used for elution, anastellin was spread out over a large number of fractions and had to be concentrated with a Centricon-10 (Millipore) as described previously (19). Anastellin (with a His 6 tag) from the pQE-70 vector (20) was also recloned into pET11b for expression in BL21(DE3) to improve solubility. However, the majority of anastellin was still insoluble and required renaturation.…”

The First Type III Repeat in Fibronectin Activates an Inflammatory Pathway in Dermal Fibroblasts

“…Preparation of the Recombinant Fibronectin Modules-All recombinant His-tagged fibronectin modules, III-1c, III-13, and III-10n, were prepared and purified as described previously (13,17,18). Briefly, PCR products were obtained by amplification of human fibronectin cDNA and cloned into bacterial expression vectors pQE-70 (III-1c and III-13) and pQE-30 (III-10n) in-frame with a bacterial His 6 tag (Qiagen, Inc., Valencia, CA).…”

Conformational remodeling of the fibronectin matrix selectively regulates VEGF signaling

“…The cryptic polymerization activity present in the III 1 domain of fibronectin can be recapitulated in a peptide termed anastellin (Morla et al, 1994;Morla and Ruoslahti, 1992). Subsequent studies showed that anastellin can bind directly to previously assembled fibronectin matrix and alter its conformation (Klein et al, 2003;Prabhakaran et al, 2009). Anastellin has been found to inhibit tumor angiogenesis in vivo as well as endothelial cell proliferation in vitro; however, its mechanism of action has remained elusive (Ambesi et al, 2005;Yi and Ruoslahti, 2001).…”