Osteoclasts are unique cells that utilize podosomes instead of focal adhesions for matrix attachment and cytoskeletal remodeling during motility. We have shown that osteopontin (OP) binding to the αvβ3 integrin of osteoclast podosomes stimulated cytoskeletal reorganization and bone resorption by activating a heteromultimeric signaling complex that includes gelsolin, pp60c-src, and phosphatidylinositol 3′-kinase. Here we demonstrate that gelsolin deficiency blocks podosome assembly and αvβ3-stimulated signaling related to motility in gelsolin-null mice. Gelsolin-deficient osteoclasts were hypomotile due to retarded remodeling of the actin cytoskeleton. They failed to respond to the autocrine factor, OP, with stimulation of motility and bone resorption. Gelsolin deficiency was associated with normal skeletal development and endochondral bone growth. However, gelsolin-null mice had mildly abnormal epiphyseal structure, retained cartilage proteoglycans in metaphyseal trabeculae, and increased trabecular thickness. With age, the gelsolin-deficient mice expressed increased trabecular and cortical bone thickness producing mechanically stronger bones. These observations demonstrate the critical role of gelsolin in podosome assembly, rapid cell movements, and signal transduction through the αvβ3 integrin.
Rho plays a regulatory role in the formation of actin stress fibers and focal adhesions, and it is also involved in integrin-mediated signaling events. To study the role of Rho in ␣ v  3 /gelsolin-dependent signaling, the HIV-Tat peptide, hemagglutinin (HA)-tagged Rho Val-14 (constitutively active) and Rho Asn-19 (dominant negative) were transduced into avian osteoclasts. Protein transduction by HA-Tat was highly efficient, and 90 -100% of the cells were transduced with HA-tagged proteins. We demonstrate here that Rho Val-14 transduction (100 nM) stimulated gelsolin-associated phosphatidylinositol 3-kinase activity, podosome assembly, stress fiber formation, osteoclast motility, and bone resorption, mimicking osteoclast stimulation by osteopontin/␣ v  3. The effects of Rho Val-14 transduction stimulation was time-dependent. C3 exoenzyme blocked the effects of RhoVal-14 and induced podosome disassembly, loss of motility, and inhibition of bone resorption. Transduction of Rho Asn-19 produced podosome disassembly, and blocked osteopontin stimulation. These data demonstrate that integrin-dependent activation of phosphoinositide synthesis, actin stress fiber formation, podosome reorganization for osteoclast motility, and bone resorption require Rho stimulation.
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.
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