Rho-dependent Rho Kinase Activation Increases CD44 Surface Expression and Bone Resorption in Osteoclasts

Abstract

Osteoclasts from osteopontin-efficient mice exhibit decreased CD44 surface expression. Osteopontin (OPN)/ ␣ v ␤ 3 generated Rho signaling pathway is required for the surface expression of CD44. In this work we show the Rho effector, Rho kinase (ROK-␣), to be a potent activator of CD44 surface expression. ROK-␣ activation was associated with autophosphorylation, leading to its translocation to the plasma membrane, as well as its association with CD44. ROK-␣ promoted CD44 surface expression through phosphorylation of CD44 and ezrinradixin-moesin (ERM) proteins and CD44⅐ERM⅐actin complex formation. Osteoclasts from OPN؊/؊ mice exhibited an ϳ55-60% decrease in basal level ROK-␣ phosphorylation as compared with wild type osteoclasts. Furthermore, RhoVal-14 transduction was only partially effective in stimulating ROK-␣/CD44 phosphorylation, as well as CD44 surface expression, in these osteoclasts. Studies on the inhibition of Rho by C3 transferase or ROK-␣ by the specific inhibitor, Y-27632, showed a decrease in the phosphorylation mediated by ROK-␣ and CD44 surface expression. Neutralizing antibodies to ␣ v , ␤ 3 , or CD44 inhibited the migration and bone resorption of wild type osteoclasts. However, only anti-␣ v or -␤ 3 antibodies blocked OPN-induced phosphorylation of ROK-␣, CD44, and the ERM proteins. Our results strongly suggest a role for ROK-␣ in ␣ v ␤ 3 -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 ␣ v ␤ 3 and CD44 for osteoclast motility and bone resorption.Osteopontin (OPN), 1 a major component of protein complexes organized around mineralization foci, is a phosphorylated acidic glycoprotein synthesized by many cell types throughout the body in response to stress or injury (1-3). Although osteopontin serves many functions in bone cells besides regulating mineralization of the matrix, regulation of osteoclast motility and resorption is perhaps its most important. Osteopontin is a ligand for several cell surface adhesion receptors, such as, and ␣ 9 ␤ 1 (reviewed in Refs. 4 and 5). The hyaluronic acid receptor, CD44, is also a receptor for OPN (6). CD44 has been implicated in chemotaxis mediated by OPN (6, 7), as well as in bone resorption (8). CD44 expression was reported in osteoclasts of human and rat bone sections (9 -11). Weber et al. (6), have shown that the binding of soluble OPN to fibroblasts transfected with CD44 cDNA is blocked by anti-CD44 antibody. Moreover, anti-CD44 or anti-OPN antibodies inhibited the migration of CD44-transfected fibroblasts toward OPN in Boyden chambers (7), suggesting CD44-OPN interaction and a role in chemotactic function. Expression of OPN and CD44 was increased in migrating fetal fibroblasts (12), and the colocalization of CD44 and OPN was prominent at the leading edge of migrating osteoclast...

Role of the Rhoa/Rho Kinase System in Flow-Related Remodeling of Rat Mesenteric Small Arteries in Vivo

“…4b). Many putative modulators of RhoA/Rho kinase are regulated within 24 h. These include the Rho guanine nucleotide-exchange factor (Rho GEF) splice variant NET1A, the 82-kD subunit of soluble guanylate cyclase [37], the ECM molecules osteopontin [38]and tenascin [39], TGFβ3 [40], and several receptors that signal through RhoA/Rho kinase (MCP-1 receptor [41], and CSF receptor [42]) (fig. 4).…”

Osteopontin

“…67 In addition, growing evidence suggests that OPN is a major regulator of CD44 surface expression, especially in osteoclasts. 22,68,69 The in vivo relevance of OPN fragments is unclear and largely unexplored. To date, only 3 studies have addressed the role of the cryptic SVVYGLR sequence in vivo.…”

Osteopontin attenuation of dextran sulfate sodium-induced colitis in mice

“…The intracellular form of OPN has a peri-membranous distribution, and it colocalizes with CD44 and ezrin-radixin-moesin proteins in migrating embryonic fibroblasts, activated macrophages, and metastatic breast cancer cells. [56][57][58] Impaired chemotaxis in OPN À/À macrophages 18 and osteoclasts 59 has been associated with reduced cell surface expression of CD44. Moreover, although expression of OPN is evidently required for the recruitment of CD44 to the cell surface in macrophages and osteoclasts, [58][59][60] this requirement is not evident in neutrophils.…”

“…[56][57][58] Impaired chemotaxis in OPN À/À macrophages 18 and osteoclasts 59 has been associated with reduced cell surface expression of CD44. Moreover, although expression of OPN is evidently required for the recruitment of CD44 to the cell surface in macrophages and osteoclasts, [58][59][60] this requirement is not evident in neutrophils. 61 The ligation of CD44 by OPN mediates chemotaxis and adhesion of fibroblasts 62 and the downregulation of interleukin-10 (IL)-10 expression in peritoneal macrophages.…”