Osteopontin (OPN) was expressed in murine wild-type osteoclasts, localized to the basolateral, clear zone, and ruffled border membranes, and deposited in the resorption pits during bone resorption. The lack of OPN secretion into the resorption bay of avian osteoclasts may be a component of their functional resorption deficiency in vitro. Osteoclasts deficient in OPN were hypomotile and exhibited decreased capacity for bone resorption in vitro. OPN stimulated CD44 expression on the osteoclast surface, and CD44 was shown to be required for osteoclast motility and bone resorption. Exogenous addition of OPN to OPN-/- osteoclasts increased the surface expression of CD44, and it rescued osteoclast motility due to activation of the alpha(v)beta(3) integrin. Exogenous OPN only partially restored bone resorption because addition of OPN failed to produce OPN secretion into resorption bays as seen in wild-type osteoclasts. As expected with these in vitro findings of osteoclast dysfunction, a bone phenotype, heretofore unappreciated, was characterized in OPN-deficient mice. Delayed bone resorption in metaphyseal trabeculae and diminished eroded perimeters despite an increase in osteoclast number were observed in histomorphometric measurements of tibiae isolated from OPN-deficient mice. The histomorphometric findings correlated with an increase in bone rigidity and moment of inertia revealed by load-to-failure testing of femurs. These findings demonstrate the role of OPN in osteoclast function and the requirement for OPN as an osteoclast autocrine factor during bone remodeling.

Parathyroid hormone (PTH), via activation of PKC and/or protein kinase A, inhibits renal proximal tubular phosphate reabsorption by facilitating the internalization of the major sodium-dependent phosphate transporter, Npt2a. Herein, we explore the hypothesis that the effect of PTH is mediated by phosphorylation of serine 77 (S77) of the first PDZ domain of the Npt2a-binding protein sodium-hydrogen exchanger regulatory factor-1 (NHERF-1). Using recombinant polypeptides representing PDZ I, S77 of NHERF-1 is phosphorylated by PKC but not PKA. When expressed in primate kidney epithelial cells (BSC-1 cells), however, activation of either protein kinase phosphorylates S77, suggesting that the phosphorylation of PDZ I by PKC and PKA proceeds by different biochemical pathways. PTH and other activators of PKC and PKA dissociate NHERF-1/ Npt2a complexes, as assayed using quantitative coimmunoprecipitation, confocal microscopy, and sucrose density gradient ultracentrifugation in mice. Murine NHERF-1 -/-renal proximal tubule cells infected with adenovirus-GFP-NHERF-1 containing an S77A mutation showed significantly increased phosphate transport compared with a phosphomimetic S77D mutation and were resistant to the inhibitory effect of PTH compared with cells infected with wild-type NHERF-1. These results indicate that PTH-mediated inhibition of renal phosphate transport involves phosphorylation of S77 of the NHERF-1 PDZ I domain and the dissociation of NHERF-1/Npt2a complexes. IntroductionParathyroid hormone (PTH) increases the urinary excretion of phosphate by facilitating the retrieval and internalization of Npt2a, the major sodium-dependent phosphate transporter found in the apical membrane of the cells of the renal proximal convoluted tubule (1-3). The precise physiologic and biochemical pathways relating activation of the PTH receptor to the endocytosis of Npt2a, however, are not known. An insight into this process was provided by the observations that Npt2a binds to the PDZ domain adaptor protein sodium-hydrogen exchanger regulatory factor-1 (NHERF-1) and that NHERF-1 -/-mice demonstrate phosphaturia and mistargeting of Npt2a (4, 5). Subsequent experiments demonstrated that NHERF-1 functions as a membrane retention signal for Npt2a and that sodium-dependent phosphate transport in renal proximal tubule cells from NHERF-1 mice was resistant to the inhibitory effect of PTH (3, 6, 7). NHERF-1 -/-cells were also resistant to the inhibitory effect of activators of PKC and PKA, the 2 major signaling pathways of the PTH1 receptor, indicating that the resistance to PTH derived

Podosomes are adhesion structures in osteoclasts and are structurally related to focal adhesions mediating cell motility during bone resorption. Here we show that gelsolin coprecipitates some of the focal adhesion-associated proteins such as c-Src, phosphoinositide 3-kinase (PI3K), p130Cas , focal adhesion kinase, integrin ␣ v ␤ 3 , vinculin, talin, and paxillin. These proteins were inducibly tyrosine-phosphorylated in response to integrin activation by osteopontin. Previous studies have defined unique biochemical properties of gelsolin related to phosphatidylinositol 3,4,5-trisphosphate in osteoclast podosomes, and here we demonstrate phosphatidylinositol 3,4,5-trisphosphate/gelsolin function in mediating organization of the podosome signaling complex. Overlay and GST pull-down assays demonstrated strong phosphatidylinositol 3,4,5-trisphosphate-PI3K interactions based on the Src homology 2 domains of PI3K. Furthermore, lipid extraction of lysates from activated osteoclasts eliminated interaction between gelsolin, cSrc, PI3K, and focal adhesion kinase despite equal amounts of gelsolin in both the lipid-extracted and unextracted experiment. The cytoplasmic protein tyrosine phosphatase (PTP)-proline-glutamic acid-serine-threonine amino acid sequences (PEST) was also found to be associated with gelsolin in osteoclast podosomes and with stimulation of ␣ v ␤ 3 -regulated phosphorylation of PTP-PEST. We conclude that gelsolin plays a key role in recruitment of signaling proteins to the plasma membrane through phospholipid-protein interactions and by regulation of their phosphorylation status through its association with PTP-PEST. Because both gelsolin deficiency and PI3K inhibition impair bone resorption, we conclude that phosphatidylinositol 3,4,5-trisphosphate-based protein interactions are critical for osteoclast function.Osteoclasts are multinucleated giant cells with bone-resorbing activity. As osteoclasts crawl over bone surfaces, they require rapid attachment and release from the extracellular matrix. Adhesion structures called podosomes present in highly motile cells are also found in osteoclasts. Osteoclasts are unique because they use the speed of podosome assembly and disassembly to generate high rates of motility. Podosome formation stabilizes the bone matrix-cell interface and forms an isolated compartment between the ruffled border and the bone surface (1, 2). Consistent with their function as adhesion sites, podosomes contain many of the same proteins found in focal adhesions, such as F-actin, vinculin, talin, gelsolin, fimbrin, and ␣-actinin (3-7).We have shown previously that osteopontin (OPN) 1 binding to integrin ␣ v ␤ 3 in osteoclasts stimulates gelsolin-associated PI3K. This leads to increased levels of gelsolin-associated polyphosphoinositides, such as phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P 2 ), phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate, uncapping of actin barbed ends, and actin filament formation (8). Moreover, OPN stimulates gelsolin-associ...

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...

Sodium-dependent phosphate transport in NHERF-1−/− proximal tubule cells does not increase when grown in a low phosphate media and is resistant to the normal inhibitory effects of parathyroid hormone (PTH). The current experiments employ adenovirus-mediated gene transfer in primary cultures of mouse proximal tubule cells from NHERF-1 null mice to explore the specific role of NHERF-1 on regulated Npt2a trafficking and sodium-dependent phosphate transport. NHERF-1 null cells have decreased sodium-dependent phosphate transport compared with wild-type cells. Infection of NHERF-1 null cells with adenovirus-GFP-NHERF-1 increased phosphate transport and plasma membrane abundance of Npt2a. Adenovirus-GFP-NHERF-1 infected NHERF-1 null proximal tubule cells but not cells infected with adenovirus-GFP demonstrated increased phosphate transport and Npt2a abundance in the plasma membrane when grown in low phosphate (0.1 mM) compared with high phosphate media (1.9 mM). PTH inhibited phosphate transport and decreased Npt2a abundance in the plasma membrane of adenovirus-GFP-NHERF-1-infected NHERF-1 null proximal tubule cells but not cells infected with adenovirus-GFP. Interestingly, phosphate transport is inhibited by activation of protein kinase A and protein kinase C in wild-type proximal tubule cells but not in NHERF-1−/− cells. Together, these results highlight the requirement for NHERF-1 for physiological control of Npt2a trafficking and suggest that the Npt2a/NHERF-1 complex represents a unique PTH-responsive pool of Npt2a in renal microvilli.