Rho-dependent Rho kinase activation increases CD44 surface expression and bone resorption in osteoclasts.
Osteoclasts from osteopontin-deficient mice exhibit decreased CD44 surface expression [corrected]. Osteopontin (OPN)/alphavbeta3 generated Rho signaling pathway is required for the surface expression of CD44. In this work we show the Rho effector, Rho kinase (ROK-alpha), to be a potent activator of CD44 surface expression. ROK-alpha activation was associated with autophosphorylation, leading to its translocation to the plasma membrane, as well as its association with CD44. ROK-alpha promoted CD44 surface expression through phosphorylation of CD44 and ezrin-radixin-moesin (ERM) proteins and CD44.ERM.actin complex formation. Osteoclasts from OPN-/- mice exhibited an approximately 55-60% decrease in basal level ROK-alpha phosphorylation as compared with wild type osteoclasts. Furthermore, RhoVal-14 transduction was only partially effective in stimulating ROK-alpha/CD44 phosphorylation, as well as CD44 surface expression, in these osteoclasts. Studies on the inhibition of Rho by C3 transferase or ROK-alpha by the specific inhibitor, Y-27632, showed a decrease in the phosphorylation mediated by ROK-alpha and CD44 surface expression. Neutralizing antibodies to alphav, beta3, or CD44 inhibited the migration and bone resorption of wild type osteoclasts. However, only anti-alphav or -beta3 antibodies blocked OPN-induced phosphorylation of ROK-alpha, CD44, and the ERM proteins. Our results strongly suggest a role for ROK-alpha in alphavbeta3-mediated Rho signaling, which is required for the phosphorylation events and CD44 surface expression. The functional deficiencies in the Rho effector(s) because of the lack of OPN were associated with decreased CD44 surface expression and hypomotility in the OPN-/- osteoclasts. Finally, we find that cooperativity exists between alphavbeta3 and CD44 for osteoclast motility and bone resorption.
Department of Oral Craniofacial Biological Sciences, University of Maryland, Baltimore, Maryland 21201, USA. mac001@dental.umaryland.edu
Meenakshi A
MA
Chellaiah
Rajat S
RS
Biswas
Susan R
SR
Rittling
David T
DT
Denhardt
Keith A
KA
Hruska
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001854:Bone Marrow Cells; D001862:Bone Resorption; D002478:Cells, Cultured; D004789:Enzyme Activation; D005434:Flow Cytometry; D018960:Hyaluronan Receptors / Q000032:analysis* / Q000378:metabolism; D007163:Immunosorbent Techniques; D039302:Integrin alphaVbeta3 / Q000276:immunology / Q000502:physiology; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010010:Osteoclasts / Q000737:chemistry / Q000502:physiology*; D053495:Osteopontin; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D012795:Sialoglycoproteins / Q000172:deficiency / Q000235:genetics / Q000494:pharmacology; D015398:Signal Transduction; D014162:Transfection; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D054460:rho-Associated Kinases
D000906:Antibodies; D018960:Hyaluronan Receptors; D039302:Integrin alphaVbeta3; D047908:Intracellular Signaling Peptides and Proteins; D011993:Recombinant Fusion Proteins; D012795:Sialoglycoproteins; C506896:Spp1 protein, mouse; D053495:Osteopontin; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D020741:rho GTP-Binding Proteins