Identification and immunolocalization of laminin in cartilage

Abstract

In a previous study on the role of integrins in the interaction of human chondrocytes with extracellular collagen and fibronectin (Dürr et al., (1993) Exp. Cell Res. 207, 235) we showed that chondrocytes adhere to laminin-1 (LN-1) in a beta 1-integrin-dependent manner. FACS analysis with various integrin antibodies including the monoclonal antibody GOH3 indicated the presence of the alpha 6 beta 1-laminin receptor on the chondrocyte surface. Anti-alpha 6 antibodies inhibited adhesion to the LN-1/E8 fragment, but not to whole laminin or heat-denatured Laminin-1, indicating that chondrocytes utilize at least two beta 1-integrins for laminin adhesion, one of which is alpha 6 beta 1 recognizing the LN-1/E8 fragment. The presence of alpha 6 beta 1-integrin on the chondrocyte surface also suggested the existence of laminin-like molecules in cartilage. Here we provide immunological and biochemical evidence in support of this possibility. Several polyclonal antibodies raised against laminin-1 or the LN-1/E8 fragment revealed a strong pericellular reaction in sections of human fetal epiphyseal cartilage and adult articular cartilage. In the fetal epiphysis laminin staining was most prominent in mature, large chondrocytes appearing in the secondary ossification zone, in particular, in the vicinity of invading capillary sprouts. Chondrocytes in the proliferating and hypertrophic zone of the growth plate and perichondrium cells were negative. All chondrocytes that stained for alpha 6-integrin also stained for laminin-1. A laminin-1-like molecule was extracted from hyaline cartilage with two bands migrating slightly faster than the alpha 1 and beta 1/gamma 1 subunits of laminin on SDS-gel electrophoresis. The two bands stained with anti-laminin-1 antibodies and could be immunoprecipitated with the same antibodies from metabolically labeled chondrocyte cultures. These findings suggests a role for laminin in developing cartilage and thus additional roles for laminins outside basement membranes.