Protein tyrosine kinase inhibitors increase cytosolic calcium and inhibit actin organization as resorbing activity in rat osteoclasts

Although there is evidence that protein tyrosine kinase inhibitors (PTKIs) suppress bone resorption activity, the mechanism of action of these compounds on osteoclastic bone resorption remains obscure. In the present study, we investigated the effect of PTKIs on cytosolic Ca(2+) concentration ([Ca(2+)](i)) and on the cytoskeleton in rat osteoclasts. The PTKIs, genistein and herbimycin A, reversibly elevated [Ca(2+)](i) measured by fura-2 microfluorimetry. The PTKI-induced increase was abolished by omission of extracellular Ca(2+), but was not attenuated by depletion of Ca(2+) stores. The PTKI-induced increase was inhibited by addition of La(3+) and Ni(2+), but not abolished by dihydropyridine (DHP) Ca(2+) channel blockers. Genistin, an inactive analogue of genistein, had no effect on [Ca(2+)](i). In the cytoskeleton assay, genistein rapidly disrupted the actin ring formation that serves as a marker for the resorbing state of osteoclasts. Disruption of the actin ring formation was also diminished in Ca(2+)-free extracellular solution. These results suggest that PTKIs in rat osteoclasts elevate [Ca(2+)](i) via activation of a DHP-insensitive, nonspecific Ca(2+) entry pathway and disrupt the formation of actin rings, resulting in suppression of bone resorption activity. The regulation of this Ca(2+)-influx by PTKIs is likely to contribute to inhibition of bone resorption by these compounds.     

Demonstration of bone resorbing cells in elasmobranchs: Comparison with osteoclasts

We have previously shown that Elasmobranchs-characterized by a partially calcified cartilaginous endoskeleton-presented a bony vertebral arch containing osteoblasts, osteocytes and resorbing cells. The aim of this study is to test the ability of Elasmobranchs to resorb bone tissue. The subcutaneous implantation in dogfish (Scyliorhinus canicula) of devitalized mineral-containing bone particles, obtained from a bony fish (the eel, Anguilla anguilla) resulted, after 21 2 months, in the formation of mononucleated as well as multinucleated cells around and between the bone fragments. By light microscopy, the multinucleated giant cells presented the general aspect of osteoclastic cells whereas, by transmission electron microscopy they never showed ruffled borders which are considered as the typical features of osteoclasts. Except for this character, the mononucleated and multinucleated cells exhibited the typical ultrastructural aspects leading us to say that these cells are involved in the resorption of the bone fragments. This study shows that Elasmobranchs are able to resorb implanted bone.     

Natural uranium impairs the differentiation and the resorbing function of osteoclasts

BackgroundUranium is a naturally occurring radionuclide ubiquitously present in the environment. The skeleton is the main site of uranium long-term accumulation. While it has been shown that natural uranium is able to perturb bone metabolism through its chemical toxicity, its impact on bone resorption by osteoclasts has been poorly explored. Here, we examined for the first time in vitro effects of natural uranium on osteoclasts.MethodsThe effects of uranium on the RAW 264.7 monocyte/macrophage mouse cell line and primary murine osteoclastic cells were characterized by biochemical, molecular and functional analyses.ResultsWe observed a cytotoxicity effect of uranium on osteoclast precursors. Uranium concentrations in the μM range are able to inhibit osteoclast formation, mature osteoclast survival and mineral resorption but don't affect the expression of the osteoclast gene markers Nfatc1, Dc-stamp, Ctsk, Acp5, Atp6v0a3 or Atp6v0d2 in RAW 274.7 cells. Instead, we observed that uranium induces a dose-dependent accumulation of SQSTM1/p62 during osteoclastogenesis.ConclusionsWe show here that uranium impairs osteoclast formation and function in vitro. The decrease in available precursor cells, as well as the reduced viability of mature osteoclasts appears to account for these effects of uranium. The SQSTM1/p62 level increase observed in response to uranium exposure is of particular interest since this protein is a known regulator of osteoclast formation. A tempting hypothesis discussed herein is that SQSTM1/p62 dysregulation contributes to uranium effects on osteoclastogenesis.General significanceWe describe cellular and molecular effects of uranium that potentially affect bone homeostasis.     

NADPH-oxidase expression and in situ production of superoxide by osteoclasts actively resorbing bone

Recent reports have suggested that production of superoxide or other reactive oxygen species by activated osteoclasts may play a role in the complex process of bone resorption; however, the enzyme responsible for production of superoxide by osteoclasts has not been characterized. To determine if osteoclasts express NADPH-oxidase, a superoxide-generating enzyme found in phagocytic leukocytes, immunohistochemical studies were performed on tibia from 1-5-d-old rats using mAbs 449 and 48 and an antiserum specific for p47-phox. These antibodies recognize epitopes on the alpha and beta subunits of cytochrome b558, respectively, and the p47 cytosolic component of NADPH-oxidase. We found that osteoclasts attached to bone surfaces in tibia expressed all three components, as did mature polymorphonuclear and some mononuclear leukocytes in the bone marrow. In many adherent osteoclasts, the cytochrome b558 subunits were localized to the ruffled-border and bone interfaces. Studies were also performed on mature rat tibia that had undergone controlled fracture. By two weeks the healing fractures develop a callus rich in actively resorbing osteoclasts. Osteoclasts within the calluses, and attached to bone surface, also expressed the cytochrome b558 proteins. In addition to demonstrating the expression of NADPH-oxidase, the active production of superoxide by osteoclasts was also demonstrated in situ in freshly isolated tibia using 3,3'-diaminobenzidine (DAB)-Mn2+, a histochemical method specific for superoxide localization. Osteoclasts attached to bone surfaces contained deposits of oxidized DAB which were observed by light microscopy. Nonstimulated polymorphonuclear and mononuclear leukocytes in the bone marrow did not contain DAB deposits unless stimulated with phorbol myristate acetate, a known activator of NADPH-oxidase. These findings indicate that osteoclasts contain NADPH-oxidase, and during the process of resorbing bone, are actively producing superoxide.     

The tyrosine kinase activity of c-Src regulates actin dynamics and organization of podosomes in osteoclasts

Podosomes are dynamic actin-rich structures composed of a dense F-actin core surrounded by a cloud of more diffuse F-actin. Src performs one or more unique functions in osteoclasts (OCLs), and podosome belts and bone resorption are impaired in the absence of Src. Using Src^−/− OCLs, we investigated the specific functions of Src in the organization and dynamics of podosomes. We found that podosome number and the podosome-associated actin cloud were decreased in Src^−/− OCLs. Videomicroscopy and fluorescence recovery after photobleaching analysis revealed that the life span of Src^−/− podosomes was increased fourfold and that the rate of actin flux in the core was decreased by 40%. Thus, Src regulates the formation, structure, life span, and rate of actin polymerization in podosomes and in the actin cloud. Rescue of Src^−/− OCLs with Src mutants showed that both the kinase activity and either the SH2 or the SH3 binding domain are required for Src to restore normal podosome organization and dynamics. Moreover, inhibition of Src family kinase activities in Src^−/− OCLs by Src inhibitors or by expressing dominant-negative Src^K295M induced the formation of abnormal podosomes. Thus, Src is an essential regulator of podosome structure, dynamics and organization.     

Recombinant VSV G proteins reveal a novel raft-dependent endocytic pathway in resorbing osteoclasts

Transcytotic membrane flow delivers degraded bone fragments from the ruffled border to the functional secretory domain, FSD, in bone resorbing osteoclasts. Here we show that there is also a FSD-to-ruffled border trafficking pathway that compensates for the membrane loss during the matrix uptake process and that rafts are essential for this ruffled border-targeted endosomal pathway. Replacing the cytoplasmic tail of the vesicular stomatitis virus G protein with that of CD4 resulted in partial insolubility in Triton X-100 and retargeting from the peripheral non-bone facing plasma membrane to the FSD. Recombinant G proteins were subsequently endosytosed and delivered from the FSD to the peripheral fusion zone of the ruffled border, which were both rich in lipid rafts as suggested by viral protein transport analysis and visualizing the rafts with fluorescent recombinant cholera toxin. Cholesterol depletion by methyl-beta-cyclodextrin impaired the ruffled border-targeted vesicle trafficking pathway and inhibited bone resorption dose-dependently as quantified by measuring the CTX and TRACP 5b secreted to the culture medium and by measuring the resorbed area visualized with a bi-phasic labeling method using sulpho-NHS-biotin and WGA-lectin. Thus, rafts are vital for membrane recycling from the FSD to the late endosomal/lysosomal ruffled border and bone resorption.     

Regulation of actin ring formation by rho GTPases in osteoclasts

Actin ring formation is a prerequisite for osteoclast bone resorption. Although gelsolin null osteoclasts failed to exhibit podosomes, actin ring was observed in these osteoclasts. Wiscott-Aldrich syndrome protein (WASP) was observed in the actin ring of gelsolin null osteoclast. Osteoclasts stimulated with osteopontin simulated the effects of Rho and Cdc42 in phosphatidylinositol 4,5-bisphosphate (PIP2) association with WASP as well as formation of podosomes, peripheral microfilopodia-like structures, and actin ring. To explore the potential functions of Rho and Cdc42, TAT-mediated delivery of Rho proteins into osteoclasts was performed. Although Rho and Cdc42 are required for actin ring formation, transduction of either one of the proteins alone is insufficient for this process. Addition of osteopontin to osteoclasts transduced with Cdc42Val12 or transduction of osteoclasts with both RhoVal14 and Cdc42Val12 augments the formation of WASP-Arp2/3 complex and actin ring. Neomycin, an antibiotic, blocked the effects of osteopontin or TAT-RhoVal14 on PIP2 interaction with WASP. WASP distribution was found to be cytosolic in these osteoclasts. Depletion of WASP by short interfering RNA-mediated gene silencing blocked actin polymerization as well as actin ring formation in osteoclasts. These results suggest that Rho-mediated PIP2 interaction with WASP may contribute to the activation and membrane targeting of WASP. Subsequent interaction of Cdc42 and Arp2/3 with WASP may enhance cortical actin polymerization in the process of actin ring formation in osteoclasts.     

Rotational dynamics of actin

The rotational diffusion of actin was studied with the technique of time-resolved phosphorescence anisotropy using actin labeled at Cys-374 with erythrosin iodoacetamide. Immediately after the polymerization of actin was initiated, the correlation time increased sharply, passing through a maximum at 5 min and then declined to low values. F-Actin at equilibrium showed no anisotropy decay. The results were interpreted as indicating the initial formation of short mobile filaments which became increasingly immobile as elongation proceeded, leaving a decay which was dominated by shorter filaments. Some of these short filaments could have arisen by fragmentation of longer filaments. Eventually, the shorter filaments themselves became immobilized by entanglement within the gel matrix. The infinite-time anisotropy increased during polymerization, reflecting a smaller range of angular motion of the probe brought about by restricted torsional motion on the submicrosecond time scale. The results were compared with the length distribution of actin filaments revealed by electron microscopy [Kawamura, M., & Maruyama, K. (1970) J. Biochem. (Tokyo) 67, 437-457]. Polymerization in the presence of 1 microM cytochalasin B abolished the maximum in the correlation time profile and tended to prevent the immobilization of filaments by favoring shorter capped filaments which retained considerable rotational freedom. Addition of spectrin dimer to F-actin caused an increase in the time-invariant anisotropy. Subsequent additions of spectrin-binding proteins (erythrocyte bands 2.1 and 4.1) caused further increases in the anisotropy in a concentration-dependent manner, suggesting additional restriction of submicrosecond torsional motions. The results suggest that actin filaments within nonmuscle cells are rotationally immobile particularly if they are cross-linked by actin-binding proteins.     

Ultrastructural and lanthanum tracer examination of rapidly resorbing rat alveolar bone suggests that osteoclasts internalize dying bone cells

Glutaraldehyde-formaldehyde fixed undecalcified alveolar bone from 7-day-old rats was prepared for light and electron microscopy. Colloidal lanthanum was used as an ultrastructural tracer, and both random and semi-serial sections were examined. Lanthanum penetrated the infoldings of the ruffled border and some nearby vacuoles and vesicles. The majority of vacuoles and vesicles were lanthanum-free. Some osteoclast profiles contained a large vacuole with a cell enclosed in its interior. The enclosed cell exhibited an irregular nucleus containing condensed peripheral chromatin, intact cytoplasmic organelles, conspicuous rough endoplasmic reticulum and large blebs on the cell surface. These features are characteristic of osteoblasts or bone-lining cells or immature osteocytes which may be undergoing apoptosis or necrosis. The observation of remnants of cellular structures within internalized osteoclast vacuoles, together with the above results, suggests that osteoclasts engulf and probably degrade dying osteoblasts/bone-lining cells or immature osteocytes. Received: 29 April 1997 / Accepted: 20 February 1998     

Podosomes display actin turnover and dynamic self-organization in osteoclasts expressing actin-green fluorescent protein

Podosomes, small actin-based adhesion structures, differ from focal adhesions in two aspects: their core structure and their ability to organize into large patterns in osteoclasts. To address the mechanisms underlying these features, we imaged live preosteoclasts expressing green fluorescent protein-actin during their differentiation. We observe that podosomes always form inside or close to podosome groups, which are surrounded by an actin cloud. Fluorescence recovery after photobleaching shows that actin turns over in individual podosomes in contrast to cortactin, suggesting a continuous actin polymerization in the podosome core. The observation of podosome assemblies during osteoclast differentiation reveals that they evolve from simple clusters into rings that expand by the continuous formation of new podosomes at their outer ridge and inhibition of podosome formation inside the rings. This self-organization of podosomes into dynamic rings is the mechanism that drives podosomes at the periphery of the cell in large circular patterns. We also show that an additional step of differentiation, requiring microtubule integrity, stabilizes the podosome circles at the cell periphery to form the characteristic podosome belt pattern of mature osteoclasts. These results therefore provide a mechanism for the patterning of podosomes in osteoclasts and reveal a turnover of actin inside the podosome.     

Intermolecular dynamics and function in actin filaments

Structural models of F-actin suggest that three segments in actin, the DNase I binding loop (residues 38-52), the hydrophobic plug (residues 262-274) and the C-terminus, contribute to the formation of an intermolecular interface between three monomers in F-actin. To test these predictions and also to assess the dynamic properties of intermolecular contacts in F-actin, Cys-374 pyrene-labeled skeletal alpha-actin and pyrene-labeled yeast actin mutants, with Gln-41 or Ser-265 replaced with cysteine, were used in fluorescence experiments. Large differences in Cys-374 pyrene fluorescence among copolymers of subtilisin-cleaved (between Met-47 and Gly-48) and uncleaved alpha-actin showed both intra- and intermolecular interactions between the C-terminus and loop 38-52 in F-actin. Excimer band formation due to intermolecular stacking of pyrene probes attached to Cys-41 and Cys-265, and Cys-41 and Cys-374, in mutant yeast F-actin confirmed the proximity of these residues on the paired sites (to within 18 A) in accordance with the models of F-actin structure. The dynamic properties of the intermolecular interface in F-actin formed by loop 38-52, plug 262-274 and the C-terminus may account for the observed cross-linking of these sites with reagents < 18 A. The functional importance of actin filament dynamics was demonstrated by the inhibition of the in vitro motility in the Gln-41-Cys-374 cross-linked actin filaments.     

Regulation of actin cytoskeleton dynamics in cells

The dynamic remolding of the actin cytoskeleton is a critical part of most cellular activities, and malfunction of cytoskeletal proteins results in various human diseases. The transition between two forms of actin, monomeric or G-actin and filamentous or F-actin, is tightly regulated in time and space by a large number of signaling, scaffolding and actin-binding proteins (ABPs). New ABPs are constantly being discovered in the post-genomic era. Most of these proteins are modular, integrating actin binding, protein-protein interaction, membrane-binding, and signaling domains. In response to extracellular signals, often mediated by Rho family GTPases, ABPs control different steps of actin cytoskeleton assembly, including filament nucleation, elongation, severing, capping, and depolymerization. This review summarizes structure-function relationships among ABPs in the regulation of actin cytoskeleton assembly.     

Diversity of actin architecture in human osteoclasts: network of curved and branched actin supporting cell shape and intercellular micrometer-level tubes

Osteoclasts are multinucleated bone-resorbing cells with a dynamic actin cytoskeleton. Osteoclasts are derived from circulating mononuclear precursors. Confocal and stimulated emission depletion (STED) super-resolution microscopy was used to investigate peripheral blood-derived human osteoclasts cultured on glass surfaces. STED and confocal microscopy demonstrated that the actin was curved and branched, for instance, in the vicinity of membrane ruffles. The overall architecture of the curved actin network extended from the podosomes to the top of the cell. The other novel finding was that a micrometer-level tube containing actin bridged the osteoclasts well above the level of the culture glass. The actin filaments of the tubes originated from the network of curved actin often surrounding a group of nuclei. Furthermore, nuclei were occasionally located inside the tubes. Our findings demonstrated the accumulation of c-Src, cortactin, cofilin, and actin around nuclei suggesting their role in nuclear processes such as the locomotion of nuclei. ARP2/3 labeling was abundant at the substratum level of osteoclasts and in the branched actin network, where it localized to the branching points. We speculate that the actin-containing tubes of osteoclasts may provide a means of transportation of nuclei, e.g., during the fusion of osteoclasts. These novel findings can pave the way for future studies aiming at the elucidation of the differentiation of multinucleated osteoclasts.     

Coupling actin dynamics and membrane dynamics during endocytosis.

A convergence of cellular, genetic and biochemical studies supports the hypothesis that the actin cytoskeleton is coupled to endocytic processes, but the roles played by actin filaments during endocytosis are not yet clear. Recent studies have identified several proteins that may functionally link the endocytic machinery with actin filament dynamics. Three of these proteins, Abp1p, Pan1p and cortactin, are activators of actin assembly nucleated by the Arp2/3 complex, a key regulator of actin assembly in vivo. Two others, intersectin and syndapin, bind N-WASp, a potent activator of actin assembly via the Arp2/3 complex. All of these proteins also bind components of the endocytic machinery, and thus, could coordinately regulate actin assembly and trafficking events. Hip1R, an F-actin-binding protein that associates with clathrin-coated vesicles, may physically link endocytic vesicles to actin filaments. The GTPase dynamin is implicated in modulating actin filaments at specialized actin-rich structures of the cell cortex, suggesting that dynamin may regulate the organization of cortical actin filaments as well as regulate actin dynamics during endocytosis. Finally, myosin VI may generate actin-dependent forces for membrane invagination or vesicle movement during the early stages of endocytosis.     

Harnessing actin dynamics for clathrin-mediated endocytosis

Actin polymerization often occurs at the plasma membrane to drive the protrusion of lamellipodia and filopodia at the leading edge of migrating cells. A role for actin polymerization in another cellular process that involves the reshaping of the plasma membrane--namely endocytosis--has recently been established. Live-cell imaging studies are shedding light on the order and timing of the molecular events and mechanisms of actin function during endocytosis.