c-Src Is Required for Stimulation of Gelsolin-associated Phosphatidylinositol 3-Kinase

We have shown that osteopontin binding to integrin α v β 3 in osteoclasts stimulates gelsolin-associated phosphatidylinositol (PtdIns) 3-hydroxyl kinase (PI 3-kinase), leading to increased levels of gelsolin-bound PtdIns 3,4-P 2 , PtdIns 4,5-P 2 , and PtdIns 3,4,5-P 3 , uncapping of barbed end actin, and actin filament formation. Inhibition of PI 3-kinase activity by wortmannin blocks osteopontin stimulation of actin filament formation, suggesting that activation of gelsolin-associated PI 3-kinase is an important pathway in cytoskeletal regulation. To study the mechanism of gelsolin-associated PI 3-kinase activation, we analyzed anti-gelsolin immunoprecipitates for the association of protein kinases. We demonstrated that c-Src co-immunoprecipitates with gelsolin, and that osteopontin stimulates its activity. Elimination of osteopontin-stimulated Src activity associated with gelsolin through antisense oligodeoxynucleotides blocked the stimulation of PI 3-kinase activity associated with gelsolin and the gelsolin-dependent cytoskeletal reorganization induced by osteopontin, including increased F-actin levels. In addition, treatment of osteoclasts with antisense oligonucleotides to Src reduced bone resorption. Our results demonstrate that osteopontin stimulates gelsolin-associated Src, leading to increased gelsolin-associated PI 3-kinase activity and PtdIns 3,4,5-P 3 levels, which facilitate actin filament formation, osteoclast motility, and bone resorption.