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Human bone-derived cells are known to attach and spread on surfaces which have been precoated with fibronectin, but the contributions made by specific domains of the molecule have not yet been defined. Here we refer to the osteoblast-like cells as human bone cells. We have determined the relevance of separate regions of fibronectin, particularly the heparin-binding region, for the initial attachment and spreading of these cells. Human bone cells attached to fragments from each of the cell- and heparin-binding regions of fibronectin, but failed to attach to a fragment from the gelatin-binding region. Bovine corneal epithelial cells, which were included as an example of an alternative primary cell strain, attached to the cell-binding fragment but showed no specific shortterm attachment to the heparin or gelatin-binding fragments. Monoclonal antibody MAb17, which binds to the cell binding region of fibronectin, partially inhibited the attachment of both human bone cells and corneal epithelial cells to intact fibronectin when present at 50 µg/ml and reduced human bone cell attachment to the cell-binding region fragment of fibronectin. Monoclonal antibody, MAb 32, which binds to the heparin-binding region of fibronectin, failed to inhibit attachment of the human bone cells to fibronectin but reduced the attachment of these cells to the heparin-binding region fragment. Heparin and chondroitin sulphate were able to inhibit human bone cell attachment to the heparin-binding fragment of fibronectin but had no effect on their attachment to intact fibronectin or the cell-binding region of fibronectin. Immunofluorescent staining and confocal microscopy showed extensive spreading and actin filament formation when human bone cells were cultured on intact fibronectin. Cells cultured on the heparin-binding fragment showed only minimal spreading coinciding with less extensive actin filament organisation. On the cell-binding fragment of fibronectin more spreading was seen than on the heparin-binding fragment but it was not as extensive as on intact fibronectin. Taken together, these data suggest that human bone cells, unlike bovine corneal epithelial cells, have an attachment mechanism for the heparin-binding region of fibronectin. Attachment to this region is probably mediated by cell surface proteoglycans. However, interaction with the cell-binding domain is required for effective cell spreading of human bone cells on fibronectin during the first 90 minutes after seeding into culture.

The initial attachment and spreading of endothelial cells from human umbilical artery onto type i collagen, type IV collagen or gelatin substrata was shown to be enhanced by inclusion of serum in the culture medium. To test whether this serum effect was mediated by adsorption of serum fibronectin or vitronectin onto the collagen, these adhesive glycoproteins were selectively removed from the serum prior to addition to the culture medium. The stimulatory effect of serum on human endothelial cell spreading on collagens I and IV was also observed with serum from which either fibronectin or vitronectin, or both, had been selectively removed. The stimulatory effect for cell spreading on gelatin was diminished by selective removal of serum fibronectin, but unaffected by removal of vitronectin. Hu man endothelial cell attachment and spreading onto tissue culture plastic was abolished by removal of vitronectin from the serum in the culture medium. These results emphasize that the native structure of collagens is required for serum-enhancement of human endothelial cell attachment and spreading on native collagen types I and TV, and show that on these substrata the stimulated adhesion and spreading are not dependent upon adsorption of serum fibronectin or vitronectin onto the collagen substratum.

A panel of monoclonal antibodies (mAbs) to bovine fibronectin (FN) is described which modulates either heparin binding or cell adhesion to FN, or both. A combination of competitive exclusion and binding to proteolytic fragments identified epitopes in the Hep II, Hep III/l and CBF (cell binding fragment) regions of FN. mAb A17, which bound to the CBF region, strongly inhibited the cell adhesion of BHK-21 fibroblasts, primary corneal fibroblasts and endothelial cells, and NM4 mammary adenocarcinoma cells, to FN at mAh concentrations as low as 1 μg/ml. This mAh was not so effective at inhibiting the adhesion of Bib mouse melanoma cells. Adhesion of B16 cells to FN was more sensitive to inhibition by mAbs binding to Hep H (A2, A9, A32, A35). Of these, A32 and A35 significantly increased the binding of 35S-heparin to FN, whereas A2 and A9 did not affect it. mAbs A2, A9 and A32 showed good binding to HBF, the 40 kDa proteolytic fragment of human FN which contains both Hep H and IIICS (type III connecting segment). These mAbs inhibited B16 cell adhesion to the HBF (heparin binding fragment) by 3050%, the greatest inhibition being shown by mAb A32. Two synthetic peptides from the HBF, CS1 (peptide 1) from the IIICS region and peptide I from the Hep H region, also inhibited B16 cell adhesion to HBF by approximately 70 and 30%, respectively. These results suggest that maximal cell adhesion to the HBF involves both CS1 and Hep H. The inhibitory effects of the two peptides were linearly additive in combination, whereas the inhibitory mAbs A2, A9 and A32 showed synergistic additive effects with each of the peptides. This points to the existence of an additional important cell binding site in Hep H, other than peptide I. Recent independent evidence for an additional cell binding site in Hep H supports this view. Melanoma cellular receptor(s) for the Hep H region may be cell surface proteoglycans but do not appear to bind to areas of Hep H with high affinity for soluble heparin, as the latter was not an inhibitor of B16 cell adhesion to the HBF. The increased effectiveness of A32 in inhibiting cell adhesion, compared to A2 and A9, may be due to conformational effects which increase the binding of soluble heparin, but reduce affinity for the ceUular receptor. These results are discussed in context with other reports in the literature.