title: Fibronectin regulates calvarial osteoblast differentiation.
abstract: The secretion of fibronectin by differentiating osteoblasts and its accumulation at sites of osteogenesis suggest that fibronectin participates in bone formation. To test this directly, we determined whether fibronectin-cell interactions regulate progressive differentiation of cultured fetal rat calvarial osteoblasts. Spatial distributions of alpha 5 integrin subunit, fibronectin, osteopontin (bone sialoprotein I) and osteocalcin (bone Gla-protein) were similar in fetal rat calvaria and mineralized, bone-like nodules formed by cultured osteoblasts. Addition of anti-fibronectin antibodies to cultures at confluence reduced subsequent formation of nodules to less than 10% of control values, showing 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 levels for alkaline phosphatase and osteocalcin were suppressed, whereas fibronectin, type I collagen and osteopontin were unaffected. To identify functionally relevant domains of fibronectin, we treated cells with soluble fibronectin fragments and peptides. Cell-binding fibronectin fragments (type III repeats 6-10) containing the Arg-Gly-Asp (RGD) sequence blocked both nodule initiation and maturation, whether or not they 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 RGD sequence, other features of the large cell-binding fragments contribute to the full osteogenic effects of fibronectin. Nodule formation and osteoblast differentiation resumed after anti-fibronectin antibodies or GRGDSPK peptides were omitted from the media, showing that the inhibition was reversible and the treatments were not cytotoxic. Outside the central cell-binding domain, peptides from the IIICS region and antibodies to the N terminus did not inhibit nodule formation. We conclude that osteoblasts interact with the central cell-binding domain of endogenously produced fibronectin during early stages of differentiation, and that these interactions regulate both normal morphogenesis and gene expression.
authors.affiliation: Department of Stomatology, University of California, San Francisco 94143-0512, USA.
authors.forename: A M
authors.initials: AM
authors.lastname: Moursi
authors.forename: C H
authors.initials: CH
authors.lastname: Damsky
authors.forename: J
authors.initials: J
authors.lastname: Lull
authors.forename: D
authors.initials: D
authors.lastname: Zimmerman
authors.forename: S B
authors.initials: SB
authors.lastname: Doty
authors.forename: S
authors.initials: S
authors.lastname: Aota
authors.forename: R K
authors.initials: RK
authors.lastname: Globus
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D005109:Extracellular Matrix / Q000378:metabolism; D005353:Fibronectins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D015870:Gene Expression; D008954:Models, Biological; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000494:pharmacology; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D010012:Osteogenesis / Q000502:physiology; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology; D051381:Rats; D012886:Skull / Q000166:cytology / Q000378:metabolism
chemical_list: D005353:Fibronectins; D009842:Oligopeptides; D010446:Peptide Fragments; C086456:glycyl- arginyl-glycyl-aspartyl-seryl-prolyl-lysine; C047981:arginyl-glycyl-aspartic acid