A quantitative analysis of the incorporation of fibulin-1 into extracellular matrix indicates that fibronectin assembly is required.

Fibulin-1 is an extracellular matrix glycoprotein found in both loose and dense connective tissues, elastic fibers and some basement membranes. Cultured cells such as fibroblasts assemble endogenously synthesized or exogenously added fibulin-1 into matrix fibrils that also contain fibronectin. Since we have previously shown that fibulin-1 binds to fibronectin (Balbona, K., Tran, H., Godyna, S., Ingham, K. C., Strickland, D. K. and Argraves, W. S. J. Biol. Chem. 267: 20120-20125, 1992), we sought to investigate fibulin-1 incorporation into fibroblast extracellular matrix with an emphasis on evaluating the potential role of fibronectin in the process. In this study, we have used quantitative assays to measure the binding of 125I-fibulin to monolayers of cultured fibroblasts. Our results show that the kinetics of fibulin-1 incorporation into the cell layer and its partitioning into detergent-soluble and -insoluble fractions were similar to those of fibronectin. It was found that antibodies to fibronectin or to the fibulin-1-binding domain of fibronectin-inhibited fibulin-1 incorporation. Cell lines that fail to assemble fibronectin into the matrix, such as HT1080 or PFHR-9, do not incorporate fibulin-1 into their cell layers. However, when HT1080 cells were induced to assemble fibronectin by treatment with dexamethasone, they subsequently acquired the ability to incorporate fibulin-1. Moreover, treatment of cultured fibroblasts with antibodies that inhibit fibronectin assembly significantly inhibit fibulin-1 incorporation into the matrix. When increased amounts of fibronectin were incorporated into cells layers by incubating the cells for varying lengths of time with exogenous fibronectin, a corresponding increase in fibulin-1 incorporation was also observed. Taken together, the data indicate that the incorporation of fibulin-1 requires fibronectin assembly and suggests a dependence on the amount of fibronectin in a matrix. These results highlight the potential of fibronectin to control the deposition of fibulin-1 into those extracellular matrices where both proteins coincide and may have implications in the formation of fibulin-1-containing matrix structures such as basement membranes or elastic fibers.