FIBRONECTIN REGULATES CALVARIAL OSTEOBLAST DIFFERENTIATION

The secretion of fibronectin by differentiating osteoblasts and its ac cumulation at sites of osteogenesis suggest that fibronectin participa tes in bone formation. To test this directly, we determined whether fi bronectin-cell interactions regulate progressive differentiation of cu ltured fetal rat calvarial osteoblasts. Spatial distributions of alpha 5 integrin subunit, fibronectin, osteopontin (bone sialoprotein I) an d osteocalcin (bone Gla-protein) were similar in fetal rat calvaria an d mineralized, bone-like nodules formed by cultured osteoblasts. Addit ion of antifibronectin antibodies to cultures at confluence reduced su bsequent formation of nodules to less than 10% of control values, show ing that fibronectin is required for normal nodule morphogenesis. Anti -fibronectin antibodies selectively inhibited steady-state expression of mRNA for genes associated with osteoblast differentiation; mRNA lev els for alkaline phosphatase and osteocalcin were suppressed, whereas fibronectin, type I collagen and osteopontin were unaffected. To ident ify functionally relevant domains of fibronectin, we treated cells wit h soluble fibronectin fragments and peptides. Cell-binding fibronectin fragments (type III repeats 6-10) containing the Arg-Gly-Asp (RGD) se quence blocked both nodule initiation and maturation, whether or not t hey contained a functional synergy site. In contrast, addition of the RGD-containing peptide GRGDSPK alone did not inhibit nodule initiation , although it did block nodule maturation. Thus, in addition to the RG D sequence, other features of the large cell-binding fragments contrib ute to the full osteogenic effects of fibronectin. Nodule formation an d osteoblast differentiation resumed after anti-fibronectin antibodies or GRGDSPK peptides were omitted from the media, showing that the inh ibition was reversible and the treatments were not cytotoxic. Outside the central cell-binding domain, peptides from the IIICS region and an tibodies to the N terminus did not inhibit nodule formation. We conclu de that osteoblasts interact with the central cell-binding domain of e ndogenously produced fibronectin during early stages of differentiatio n, and that these interactions regulate both normal morphogenesis and gene expression.