Extracellular matrix remodeling and cell signaling by the first type III repeat in fibronectin

Matricryptic sites derived from extracellular matrix molecules have been shown to modify important biological processes such as wound repair, tumor growth and angiogenesis. One of these sites, located in fibronectin's first Type III repeat (III1c), influences extracellular matrix assembly and signaling pathways relevant to cytoskeletal organization and cell cycle progression. The purpose of this study was to identify possible mechanisms responsible for transducing 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 III 13 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 III 1c, 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 the binding of III 1c to cell layers did not result in 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, recognized by the monoclonal antibody IST-9, present within the EDA Type III module of cellular fibronectin. Kinetic analysis revealed that initial loss of the EDA epitope occurred within 6 min, with a maximal effect obtained 1 hr following the addition of III1c. Inhibition of IST-9 binding was cell independent and resulted from a direct interaction between III1c and fibronectin, although III1c did not bind preferentially to the EDA region. The III1c induced loss of the EDA epitope was associated with the activation of p38 MAPK followed by the formation of filopodia. The effect of III 1c on cell-signaling pathways and the cytoskeleton were dose- and time dependent. Maximal p38 MAPK phosphorylation occurred within 1 hr, while cytoskeletal changes did not appear until 12 hrs later. The time-course of p38 MAPK activation correlated with the phosphorylation of its upsteam kinase MKK3/6. Experiments using beta1 integrin null cells and blocking antibodies indicated that beta1 integrin were not necessary for the activation of p38 MAPK by III1c. Likewise, fibronectin was not found to be involved in the III1c activation of p38 MAPK. Cell spreading was required for complete activation of p38 MAPK in response to III1c treatment, suggesting that actin organization may be involved. 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. However, fibronectin matrix remodeling properties of III1c does not appear to mediate the effects of III1c on p38 MAPK activity. In addition, our results suggest that III1c activation of p38 MAPK is mediated through an unidentified receptor whose activity is partially dependent on actin organization.