Isoform-specific attachment of osteoprogenitors to laminins: Mapping to the short arms of laminin-1

The recruitment of osteoblast progenitors involves their migration and atta chment to the sites of bone formation through interactions with matrix prot eins. In a time-limited cell attachment assay, coated laminin-1 inhibits th e adhesion of most rat calvaria cells but attaches specifically to osteopro genitors, as quantified by the number of bone colonies (nodules) formed in the cultures. In order to determine the molecular mechanisms involved in os teoprogenitor attachment to laminin-1, we investigated the effects of lamin in-5 a N-truncated laminin variant. In contrast to laminin-1, laminin-5 inc reased (1.5-fold) rat calvaria cell attachment and did not display any spec ific affinity for osteoprogenitors. In competition experiments on laminin-5 , blocking antibodies directed against either the integrin chain beta 1 or the C-terminal portion of laminin-5, as well as thermic denaturation of the protein at 80 degrees C, inhibited rat calvaria cell attachment, suggestin g the implication of integrin alpha 3 beta 1 binding to the conformation-de pendent C-terminal end of laminin-5. Stepwise thermic denaturation did not suppress the anti-adhesive activity of laminin-1, while osteoprogenitor rec ruitment was abolished after denaturation above 60 degrees C, suggesting th at different domains are involved in these two effects. The anti-beta 1 ant ibody further decreased RC cell attachment to laminin-1, providing evidence for concomitant anti-adhesive and beta 1-dependent cell attachment activit ies. Blocking of beta 1 integrin subunit did not, however, reduce osteoprog enitor recruitment. Finally, purified elastase digestion fragment E1+, enco mpassing the N-terminal short arms of laminin-1, reproduced the effects of the complete molecule in the assay, while C-terminal fragment E8did not dis play any cell attachment or osteoprogenitor recruitment properties. In conc lusion, the anti-adhesive and osteoprogenitor-selective effects of laminin- 1 on rat calvaria cell populations are distinct, beta 1-integrin-independen t properties mapping to the short arms of the molecule and thus not display ed by the truncated laminin-5. (C) 1999 Academic Press.