Abstract
The importance of the cooperative function of osteopontin (OPN) and CD44 in cell adhesion, cell migration, immune responses and metastatic capacity of tumor cells has been confirmed recently. Our previous studies have shown that OPN colocalizes with CD44 inside embryonic fibroblasts, concanavalin A-activated human macrophages, and metastatic breast adenocarcinoma cell lines and plays a critical role in the migration of these cells. Also, we have reported that monolayer cultures of pre-fusion osteoclasts extend many pseudopods and fuse into large multinuclear osteoclasts, while these processes were suppressed in osteoclasts from the OPN-/- and CD44-/- mice. To further investigate the importance of OPN and CD44 in cell migration, especially in relation to cytoskeleton organization, we first analyzed the spatial relationship between OPN, CD44 and actin in pre-fusion osteoclasts generated from rat bone marrow monocytes. Immunofluorescence analyses showed that OPN colocalized with CD44 in the cell processes of osteoclast precursor cells. However, this colocalization was completely lost in the mature osteoclasts with actin ring. Next, we examined whether OPN influences the hyaluronan (HA) binding to CD44 in embryonic fibroblasts, using bead binding assay. In this study, the number of HA-coated beads attached to OPN-/- cells significantly reduced in comparison to OPN+/+ cells, whereas there was no difference in BSA- or fibronectin (FN) -coated bead attachment between OPN-/- and OPN+/+ cells, indicating that OPN influences the CD44 ligation by HA, but not FN. In conclusion, our present study using double-immunostaining analyses showes that OPN colocalizes with CD44 and actin in the cell processes in pre-fusion osteoclasts, while this colocalization was completely lost in the mature osteoclasts, both generated from rat bone marrow monocytes in the presence of M-CSF and RANKL. Moreover, bead binding assay indicates that OPN is critical for the CD44 binding to HA in embryonic mouse fibroblasts.
