Production of Soluble and Cell-Associated Fibronectin by Cultured Keratinocytes

Abstract

Fibronectin has been demonstrated in epithelial cell types in culture, but published studies of keratinocytes have shown patterns of fibronectin produced by cells grown in medium with serum, which contains fibronectin. Since plasma fibronectin can bind to cells in vitro, cells grown in serum-supplemented media could show artifactual patterns of cell-associated fibronectin. To study insoluble fibronectin produced by keratinocytes, we plated cells in the absence of feeder layers in medium lacking fibronectin. Medium conditioned by metabolically labeled keratinocytes was studied by immunoprecipitation and by extraction with gelatin-Sepharose. Cells grown in fibronectin-free medium were labeled using affinity-purified anti-fibronectin antibody and fluorescein-conjugated antirabbit IgG. Keratinocytes produced soluble fibronectin, since both immunoprecipitation and adsorption to gelatin-Sepharose detected 35S-methionine-labeled material which comigrated with human plasma fibronectin on sodium dodecyl sulfate polyacrylamide gels. Demonstration of insoluble, cell-associated fibronectin was enhanced in Triton X-100-extracted cells and was seen in subcellular fibrillar arrays at both physiologic and reduced Ca++ concentrations, but in intracellular locations only at physiologic Ca++ concentrations. When cells grown in 1.1 mM Ca++ were removed with Triton X-100, diffusely distributed fibrillar fibronectin remained on the surface of the coverslip. Asymmetric "tracks" of fibronectin left by sparsely plated cells suggested movement. Fibronectin is deposited by keratinocytes on the culture surface and may be modulated by culture conditions