Intracellular osteopontin is an integral component of the CD44-ERM complexinvolved in cell migration

Osteopontin (OPN) is a secreted glycoprotein with mineral- and cell-binding properties that can regulate cell activities through integrin receptors. P reviously, we identified an intracellular form of osteopontin with a perime mbranous distribution in migrating fetal fibroblasts (Zohar et al., J Cell Physiol 170:88-98, 1997). Since OPN and CD44 expression are increased in mi grating cells, we analyzed the relationship of these proteins with immunofl uorescence and confocal microscopy. A distinct co-localization of perimembr anous OPN and cell-surface CD44 was observed in fetal fibroblasts, periodon tal ligament cells, activated macrophages, and metastatic breast cancer cel ls. The co-localization of OPN and CD44 was prominent at the leading edge o f migrating fibroblasts, where OPN also co-localized with the ezrin/radixin /moesin (ERM) protein ezrin, as well as in cell processes and at attachment sites of hyaluronan-coated beads. The subcortical location of OPN in these cells was verified by cell-surface biotinylation experiments in which biot inylated CD44 and non-biotinylated OPN were isolated from complexes formed with hyaluronan-coated beads and identified with immunoblotting. That perim embranous OPN represents secreted protein internalized by endocytosis or ph agocytosis appeared to be unlikely since exogenous OPN that was added to ce ll cultures could nor be detected inside the cells. A physical association with OPN, CD44, and ERM, but not with vinculin or alpha-actin, was indicate d by immunoadsorption and immunoblotting of cell proteins in complexes extr acted from hyaluronan-coated beads. The functional significance of OPN in t his complex was demonstrated using OPN-/- and CD-/- mouse fibroblasts which displayed impaired migration and a reduced attachment to hyaluronan-coated beads. These studies indicate that OPN exists as an integral component of a hyaluronan-CD44-ERM attachment complex that is involved in the migration of embryonic fibroblasts, activated macrophages, and metastatic cells. J. C ell. Physiol. 184:118-130, 2000. (C) 2000 Wiley-Liss, Inc.