The recruitment of osteoblast progenitors involves their migration and attachment to the sites of bone formation through interactions with matrix proteins. In a time-limited cell attachment assay, coated laminin-1 inhibits the adhesion of most rat calvaria cells but attaches specifically to osteoprogenitors, as quantified by the number of bone colonies (nodules) formed in the cultures. In order to determine the molecular mechanisms involved in osteoprogenitor attachment to laminin-1, we investigated the effects of laminin-5, a N-truncated laminin variant. In contrast to laminin-1, laminin-5 increased (1.5-fold) rat calvaria cell attachment and did not display any specific affinity for osteoprogenitors. In competition experiments on laminin-5, blocking antibodies directed against either the integrin chain beta1 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, suggesting the implication of integrin alpha3beta1 binding to the conformation-dependent C-terminal end of laminin-5. Stepwise thermic denaturation did not suppress the anti-adhesive activity of laminin-1, while osteoprogenitor recruitment was abolished after denaturation above 60 degrees C, suggesting that different domains are involved in these two effects. The anti-beta1 antibody further decreased RC cell attachment to laminin-1, providing evidence for concomitant anti-adhesive and beta1-dependent cell attachment activities. Blocking of beta1 integrin subunit did not, however, reduce osteoprogenitor recruitment. Finally, purified elastase digestion fragment E1+, encompassing the N-terminal short arms of laminin-1, reproduced the effects of the complete molecule in the assay, while C-terminal fragment E8 did not display any cell attachment or osteoprogenitor recruitment properties. In conclusion, the anti-adhesive and osteoprogenitor-selective effects of laminin-1 on rat calvaria cell populations are distinct, beta1-integrin-independent properties mapping to the short arms of the molecule and thus not displayed by the truncated laminin-5.
Copyright 1999 Academic Press.