c-Src is required for stimulation of gelsolin-associated phosphatidylinositol 3-kinase.
We have shown that osteopontin binding to integrin alphav beta3 in osteoclasts stimulates gelsolin-associated phosphatidylinositol (PtdIns) 3-hydroxyl kinase (PI 3-kinase), leading to increased levels of gelsolin-bound PtdIns 3,4-P2, PtdIns 4,5-P2, and PtdIns 3, 4,5-P3, 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-P3 levels, which facilitate actin filament formation, osteoclast motility, and bone resorption.
Chellaiah
M
M
Renal Division, Barnes-Jewish Hospital, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Fitzgerald
C
C
Alvarez
U
U
Hruska
K
K
D008841:Actin Cytoskeleton / Q000648:ultrastructure; D000199:Actins / Q000378:metabolism; D000818:Animals; D015703:Antigens, CD / Q000502:physiology; D001862:Bone Resorption; D002448:Cell Adhesion; D002645:Chickens; D004789:Enzyme Activation; D018260:Gelsolin / Q000378:metabolism*; D017353:Gene Deletion; D039482:Integrin alpha5; D039661:Integrin beta3; D010010:Osteoclasts / Q000502:physiology* / Q000648:ultrastructure; D053495:Osteopontin; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010980:Platelet Membrane Glycoproteins / Q000502:physiology; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D012795:Sialoglycoproteins / Q000502:physiology*; D015398:Signal Transduction
D000199:Actins; D015703:Antigens, CD; D018260:Gelsolin; D039482:Integrin alpha5; D039661:Integrin beta3; D010980:Platelet Membrane Glycoproteins; D012333:RNA, Messenger; D012795:Sialoglycoproteins; D053495:Osteopontin; D019869:Phosphatidylinositol 3-Kinases; D016392:Proto-Oncogene Proteins pp60(c-src)