Recognition of cryptic sites in human and mouse laminins by rat osteoclasts is mediated by β3 and β1 integrins

Abstract

Laminins may be encountered by osteoclasts and their precursors in basement membranes when they migrate from periosteal vasculature during skeletal development and in pathological situations. We have examined the recognition by osteoclasts of intact laminins and their proteolytic derivatives, and analysed the mechanism of adhesion. Rat osteoclasts fail to bind intact mouse Engelbreth-Holm-Swarm (EHS) laminin (3% adhesion relative to adhesion to foetal calf serum proteins) and bind only weakly to native human placental laminin (13%) or human merosin (9%). Pepsin treatment of native mouse EHS and human laminins increased osteoclast adhesion. Rat osteoclasts adhered to mouse EHS laminin-derived P1 fragment (70%), but failed to bind the E8 fragment, which contains adhesion sites recognised by some integrins. Binding to human and mouse P1 laminins was abolished by treatment with RGD-containing peptides and required divalent cations, but not by YIGSR peptide. Combinations of monoclonal antibodies to rat β3 and ∝v integrins reduced binding to P1 fragment by 91% and to human laminin by 72%, demonstrating that the major integrin involved in rat osteoclast adhesion to proteolysed laminin is ∝vβ3. Antiserum to β1 integrin inhibited adhesion to human laminin by 40%, but to P1 fragment by only 8%; this suggests that β1 integrin(s) contribute to osteoclast adhesion to human laminin but probably not to P1 fragment. The involvement of ∝vβ3 integrin was confirmed using a recombinant human ∝vβ3 solid phase binding assay. ∝vβ3 bound to mouse P1 fragment and proteolytically digested human laminin, but not intact lamming. Binding of the ∝vβ3 receptor to both laminins was significantly inhibited by RGD peptides and by monoclonal antibody 9C9 to human β3 integrin. In conclusion, our findings demonstrate that rat osteoclasts bind to laminin only when cryptic peptide sites become functional after exposure by proteolytic cleavage, and that both β3 and, to a lesser degree, β1 integrins are involved in this process.