Proteome analysis of secreted proteins during osteoclast differentiation using two different methods: two-dimensional electrophoresis and isotope-coded affinity tags analysis with two-dimensional chromatography

Bone is maintained by two cell types, bone-forming osteoblasts and bone-resorbing osteoclasts. Osteoblasts express two factors, osteoprotegerin and receptor activator of NF-kappaB ligand (RANKL), inhibiting and promoting osteoclast differentiation, respectively. In contrast, modulators of bone resorption expressed by osteoclasts have not been so well studied enough. In the present study, we demonstrate proteome analysis of secreted proteins during osteoclast differentiation to elucidate the molecular mechanism of bone resorption and bone remodeling. To achieve this objective, we chose RAW264.7 cells with RANKL as a homogeneous osteoclast differentiation model and used two methods, two-dimensional gel electrophoresis (2-DE) and isotope-coded affinity tags (ICAT) analysis with two-dimensional liquid chromatography. We found 23 spots in 2-DE and 19 proteins in ICAT analysis which were expressed differently during osteoclast differentiation. These two methods gave us closely related but different information about proteins, suggesting they are complementary or at least supplementary methods at present. Cathepsins, osteopontin, legumain, macrophage inflammatory protein-1alpha, and other proteins were observed as up- or down-regulated proteins and are discussed in the context of osteoclast differentiation and bone resorption. In addition to confirming previous observations, this study indicates novel proteins related to osteoclast differentiation which are potential therapeutic targets for the treatment of bone diseases, such as osteoporosis.     

Human osteoclast ontogeny and pathological bone resorption

Monocytes and macrophages are capable of degrading both the mineral and organic components of bone and are known to secrete local factors which stimulate host osteoclastic bone resorption. Recent studies have shown that monocytes and macrophages, including those isolated from neoplastic and inflammatory lesions, can also be induced to differentiate into cells that show all the cytochemical and functional characteristics of mature osteoclasts, including lacunar bone resorption. Monocyte/macrophage-osteoclast differentiation occurs in the presence of osteoblasts/bone stromal cells (which express osteoclast differentiation factor) and macrophage-colony stimulating factor and is inhibited by osteoprotegerin. Various systemic hormones and local factors (e.g. cytokines, growth factors, prostaglandins) modulate osteoclast formation by controlling these cellular and humoral elements. Various pathological lesions of bone and joint (e.g. carcinomatous metastases, arthritis, aseptic loosening) are associated with osteolysis. These lesions generally contain a chronic inflammatory infiltrate in which macrophages form a significant fraction. One cellular mechanism whereby pathological bone resorption may be effected is through generation of increased numbers of bone-resorbing osteoclasts from macrophages. Production of humoral factors which stimulate mononuclear phagocyte-osteoclast differentiation and osteoclast activity is also likely to influence the extent of pathological bone resorption.     

Proteomic identification of the TRAF6 regulation of vacuolar ATPase for osteoclast function

Osteoclasts are cells specialized for bone resorption. For osteoclast activation, tumor necrosis factor receptor-associated factor 6 (TRAF6) plays a pivotal role. To find new molecules that bind TRAF6 and have a function in osteoclast activation, we employed a proteomic approach. TRAF6-binding proteins were purified from osteoclast cell lysates by affinity chromatography and their identity was disclosed by MS. The identified proteins included several heat shock proteins, actin and actin-binding proteins, and vacuolar ATPase (V-ATPase). V-ATPase, documented for a great increase in expression during osteoclast differentiation, is an important enzyme for osteoclast function; it transports proton to resorption lacunae for hydroxyapatite dissolution. The binding of V-ATPase with TRAF6 was confirmed both in vitro by GST pull-down assays and in osteoclasts by co-immunoprecipitation and confocal microscopy experiments. In addition, the V-ATPase activity associated with TRAF6 increased in osteoclasts stimulated with receptor activator of nuclear factor kappaB ligand (RANKL). Furthermore, a dominant-negative form of TRAF6 abrogated the RANKL stimulation of V-ATPase activity. Our study identified V-ATPase as a TRAF6-binding protein using a proteomics strategy and proved a direct link between these two important molecules for osteoclast function.     

The glutamate receptor antagonist MK801 modulates bone resorption in vitro by a mechanism predominantly involving osteoclast differentiation.

Recent identification in bone of transporters, receptors, and components of synaptic signaling suggests a role for glutamate in the skeleton. We investigated effects of glutamate and its antagonist MK801 on osteoclasts in vitro. Glutamate applied to patch clamped osteoclasts induced significant increases in whole-cell membrane currents (P<0.01) in the presence of the coagonist glycine. Agonist-elicited currents were significantly decreased after application of MK801 (100 microM, P<0.01), but MK801 had no effect on actin ring formation necessary for osteoclast polarization, attachment, and resorption. In cocultures of bone marrow cells and osteoblasts in which osteoclasts develop, MK801 inhibited osteoclast differentiation and reduced resorption of pits in dentine (3 to 100 microM; P<0.001). MK801 added early in the culture (for as little as 2-4 days) was as effective as addition for the entire culture period. Addition of MK801 for any time after day 7 of culture was ineffective in reducing osteoclast activity. Using rat and rabbit mature osteoclasts cultured on dentine or explants of mouse calvariae prelabeled with (45)Ca, we could not detect significant effects of MK801 on osteoclastic resorption. These data show clearly that glutamate receptor function is critical during osteoclastogenesis and suggest that glutamate is less important in regulating mature osteoclast activity.-Peet, N. M., Grabowski, P. S., Laketic-Ljubojevic, I., Skerry, T. M. The glutamate receptor antagonist MK801 modulates bone resorption in vitro by a mechanism predominantly involving osteoclast differentiation.     

Rab13 is upregulated during osteoclast differentiation and associates with small vesicles revealing polarized distribution in resorbing cells

Osteoclasts are bone-resorbing multinucleated cells that undergo drastic changes in their polarization due to heavy vesicular trafficking during the resorption cycle. These events require the precise orchestration of membrane traffic in order to maintain the unique characteristics of the different membrane domains in osteoclasts. Rab proteins are small GTPases involved in regulation of most, if not all, steps of vesicle trafficking. The investigators studied RAB genes in human osteoclasts and found that at least 26 RABs were expressed in osteoclasts. Out of these, RAB13 gene expression was highly upregulated during differentiation of human peripheral blood monocytic cells into osteoclasts. To study its possible function in osteoclasts, the investigators performed immunolocalization studies for Rab13 and various known markers of osteoclast vesicular trafficking. Rab13 localized to small vesicular structures at the superior parts of the osteoclast between the trans-Golgi network and basolateral membrane domain. Rab13 localization suggests that it is not involved in endocytosis or transcytosis of bone degradation products. In addition, Rab13 did not associate with early endosomes or recycling endosomes labeled with EEA1 or TRITC-conjugated transferrin, respectively. Its involvement in glucose transporter traffic was excluded as well. It is suggested that Rab13 is associated with a putative secretory function in osteoclasts.     

Artemisinin inhibits breast cancer-induced osteolysis by inhibiting osteoclast formation and breast cancer cell proliferation

In addition to being used to treat malaria, artemisinin (Art) can be used as an anti-inflammatory and antitumor agent. In this study, we evaluated the effects of Art on osteoclast formation and activation and on the development of breast cancer cells in bone. To evaluate the effect of Art on osteoclast differentiation in vitro, we treated bone marrow-derived macrophages (BMMs) with various concentrations of Art and evaluated the expression of genes and proteins involved in osteoclast formation. We also performed cell counting kit-8 assays to evaluate the toxicity of Art in BMMs and MDA-MB-231 cells. We also performed Transwell assays, wound-healing assays, colony formation assays, and cell apoptosis assays to evaluate the effect of Art in MDA-MB-231 cells. We also evaluated the effect of Art in an in vivo osteoclast bone resorption assay using a nude mouse model. We demonstrated that Art inhibits the differentiation and establishment of osteoclasts even though Art is not toxic to osteoclasts. In addition, Art reduced expression of genes involved in osteoclast formation and inhibited osteoclast bone resorption in a concentration-dependent manner. Based on our data, we believe that Art can inhibit proliferation of breast cancer cells by activating apoptosis pathways, and inhibit osteoclast formation and differentiation by inhibiting activation of cathepsin K, ATPase H+ transporting V0 subunit D2, nuclear factor of activated T cells 1, calcitonin receptor, and tartrate-resistant acid phosphatase and by inhibiting nuclear factor-κB activation.     

The Rho GTPase Wrch1 regulates osteoclast precursor adhesion and migration

An excess of osteoclastic bone resorption relative to osteoblastic bone formation results in progressive bone loss, characteristic of osteoporosis. Understanding the mechanisms of osteoclast differentiation is essential to develop novel therapeutic approaches to prevent and treat osteoporosis. We showed previously that Wrch1/RhoU is the only RhoGTPase whose expression is induced by RANKL during osteoclastogenesis. It associates with podosomes and the suppression of Wrch1 in osteoclast precursors leads to defective multinucleated cell formation. Here we further explore the functions of this RhoGTPase in osteoclasts, using RAW264.7 cells and bone marrow macrophages as osteoclast precursors. Suppression of Wrch1 did not prevent induction of classical osteoclastic markers such as NFATc1, Src, TRAP (Tartrate-Resistant Acid Phosphatase) or cathepsin K. ATP6v0d2 and DC-STAMP, which are essential for fusion, were also expressed normally. Similar to the effect of RANKL, we observed that Wrch1 expression increased osteoclast precursor aggregation and reduced their adhesion onto vitronectin but not onto fibronectin. We further found that Wrch1 could bind integrin ß3 cytoplasmic domain and interfered with adhesion-induced Pyk2 and paxillin phosphorylation. Wrch1 also acted as an inhibitor of M-CSF-induced prefusion osteoclast migration. In mature osteoclasts, high Wrch1 activity inhibited podosome belt formation. Nevertheless, it had no effect on mineralized matrix resorption. Our observations suggest that during osteoclastogenesis, Wrch1 potentially acts through the modulation of αvß3 signaling to regulate osteoclast precursor adhesion and migration and allow fusion. As an essential actor of osteoclast differentiation, the atypical RhoGTPase Wrch1/RhoU could be an interesting target for the development of novel antiresorptive drugs.     

Podosomes are dispensable for osteoclast differentiation and migration

Podosomes are adhesion structures characteristic of the myeloid cell lineage, encompassing osteoclasts, dendritic cells and macrophages. Podosomes are actin-based structures that are dynamic and capable of self-organization. In particular in the osteoclast, podosomes densely pack into a thick ring called the sealing zone. This adhesion structure is typical of osteoclasts and necessary for the resorption of the bone matrix. We thought to explore in more details the role of podosomes during osteoclast differentiation and migration. To this end, we made from soft to stiff substrates that had not been functionalized with extracellular matrix proteins. Such substrates did not support podosome formation in osteoclasts. With such devices, we could show that integrin activation was sufficient to drive podosome assembly, in a substrate stiffness independent fashion. We additionally report here that osteoclast differentiation is a podosome-independent process. Finally, we show that osteoclasts devoid of podosomes can migrate efficiently. Our study further illustrates the great capacity of myeloid cells to adapt to the different environments they encounter during their life cycle.     

Regulation of osteoclast activity.

Osteoclasts are the primary cell type responsible for bone resorption. This paper reviews many of the known regulators of osteoclast activity, including hormones, cytokines, ions, and arachidonic acid metabolites. Most of the hormones and cytokines that inhibit osteoclast activity act directly on the osteoclasts. In contrast, most of the hormones and cytokines that stimulate osteoclast activity act indirectly through osteoblasts. Particularly interesting in this regard are agents that directly inhibit activity of highly purified osteoclasts yet stimulate activity of osteoclasts that are co-cultured with osteoblasts. Recent studies have demonstrated that the primary mechanism by which bone resorptive agents stimulate osteoclast activity indirectly is likely to be up-regulation of production of osteoclast differentiation factor/osteoprotegerin ligand (ODF/OPGL) by the osteoblasts. In addition to discussing regulators of osteoclast activity per se, this paper also reviews the role of osteoclast apoptosis to limit the extent of bone resorption.     

Src kinase activity is essential for osteoclast function

Deletion of the c-src gene impairs osteoclast bone resorbing activity, causing osteopetrosis. Although it has been concluded that restoring only the Src adaptor function at least partly rescues the cell attachment and skeletal phenotypes, the contribution of Src kinase activity remains controversial. Src forms a complex with Pyk2 and Cbl after adhesion-induced stimulation of alpha(V)beta(3) integrin. To demonstrate the importance of the Pyk2-Src association in osteoclasts and to distinguish the contributions of the Src adaptor and kinase activities in cytoskeletal organization and osteoclast function, we expressed mutants of Src and Pyk2 in osteoclasts using adenovirus vectors. Eliminating the Src-binding site on Pyk2 (Pyk2(Y402F)) markedly inhibited bone resorption by osteoclast-like cells, whereas kinase-dead Pyk2 had little effect. Kinase-dead Src, unlike kinase-dead Pyk2, markedly inhibited the bone-resorbing activity of wild type osteoclasts and failed to significantly restore bone-resorbing activity to Src(-/-) osteoclast-like cells. Activation of Src kinase by overexpressing kinase-dead Csk failed to reverse the inhibitory effect of Pyk2(Y402F), suggesting that osteoclastic bone resorption requires both c-Src kinase activity and the targeting of Src kinase by Pyk2. Src-catalyzed phosphorylation of Cbl on Tyr-731 is reported to induce the activation and recruitment of phosphatidylinositol 3-kinase to the cell membrane in a signaling pathway that is critical for osteoclast function. Expressing the Cbl(Y731F) mutant in osteoclasts markedly reduced their bone resorbing activity, suggesting that phosphorylation of Cbl(Y731) and the subsequent recruitment and activation of phosphatidylinositol 3-kinase may be critical signaling events downstream of Src in osteoclasts.     

Structure and function of V-ATPases in osteoclasts: potential therapeutic targets for the treatment of osteolysis

Excessive activity of osteoclasts becomes manifest in many common lytic bone disorders such as osteoporosis, Paget's disease, bone aseptic loosening and tumor-induced bone destruction. Vacuolar proton pump H+-adenosine triphosphatases (V-ATPases), located on the bone-apposed plasma membrane of the osteoclast, are imperative for the function of osteoclasts, and thus are a potential molecular target for the development of novel anti-resorptive agents. To date, the V-ATPases core structure has been well modeled and consists of two distinct functional domains, the V1 (A, B1, B2, C1, C2, D, E1, E2, F, G1, G2, G3, and H subunits) and V0 (a1, a2, a3, a4, d1, d2, c, c' e1, e2 subunits) as well as the accessory subunits ac45 and M8-9. However, the exact configuration of osteoclast specific V-ATPases remains to be established. Inactivation of subunit a3 leads to osteopetrosis in both mice and man because of non-functional osteoclasts that are capable of acidifying the extracellular resorption lacuna. On the other hand, inactivation of subunits c, d1 and ac45 results in early embryonic lethality, indicating that certain subunits, such as a3, are more specific to osteoclast function than others. In osteoclasts, V-ATPases also cooperate with chloride channel protein CLC-7 to acidify the resorption lacuna. In addition, development of V-ATPases inhibitors such as bafilomycin A1, SB 242784 and FR167356 that selectively target osteoclast specific V-ATPases remains a challenge. Understanding the molecular and cellular mechanisms by which specific subunits of V-ATPase regulate osteoclast function might facilitate the development of novel and selective inhibitors for the treatment of lytic bone disorders. This review summarizes recent research developments in V-ATPases with particular emphasis on osteoclast biology.     

Bone matrix regulates osteoclast differentiation and annexin A8 gene expression

While attachment to bone is required for optimal osteoclast function, the molecular events that underlie this fact are unclear, other than that the cell requires adhesion to mineralized matrix to assume a fully differentiated phenotype. To address this issue, we cultured murine bone marrow-derived osteoclasts on either cell culture plastic or devitalized mouse calvariae to identify the distinct genetic profile induced by interaction with bone. Among a number of genes previously unknown to be expressed in osteoclasts we found that Annexin A8 (AnxA8) mRNA was markedly up-regulated by bone. AnxA8 protein was present at high levels in osteoclasts present in human tissues recovered from sites of pathological bone loss. The presence of bone mineral was required for up-regulation of AnxA8 mRNA since osteoclasts plated on decalcified bone express AnxA8 at low levels as did osteoclasts plated on native or denatured type I collagen. Finally, AnxA8-regulated cytoskeletal reorganization in osteoclasts generated on a mineralized matrix. Thus, we used a novel approach to define a distinct bone-dependent genetic program associated with terminal osteoclast differentiation and identified Anxa8 as a gene strongly induced late in osteoclast differentiation and a protein that regulates formation of the cell's characteristic actin ring.     

Osteoprotegerin inhibit osteoclast differentiation and bone resorption by enhancing autophagy via AMPK/mTOR/p70S6K signaling pathway in vitro

Osteoclasts are highly differentiated terminal cells formed by fusion of hematopoietic stem cells. Previously, osteoprotegerin (OPG) inhibit osteoclast differentiation and bone resorption by blocking receptor activator of nuclear factor-κB ligand (RANKL) binding to RANK indirect mechanism. Furthermore, autophagy plays an important role during osteoclast differentiation and function. However, whether autophagy is involved in OPG-inhibited osteoclast formation and bone resorption is not known. To elucidate the role of autophagy in OPG-inhibited osteoclast differentiation and bone resorption, we used primary osteoclast derived from mice bone marrow monocytes/macrophages (BMM) by induced M-CSF and RANKL. The results showed that autophagy-related proteins expression were upregulated; tartrate-resistant acid phosphatase-positive osteoclast number and bone resorption activity were decreased; LC3 puncta and autophagosomes number were increased and activated AMPK/mTOR/p70S6K signaling pathway. In addition, chloroquine (as the autophagy/lysosome inhibitor, CQ) or rapamycin (as the autophagy/lysosome inhibitor, Rap) attenuated osteoclast differentiation and bone resorption activity by OPG treatment via AMPK/mTOR/p70S6K signaling pathway. Our data demonstrated that autophagy plays a critical role in OPG inhibiting osteoclast differentiation and bone resorption via AMPK/mTOR/p70S6K signaling pathway in vitro.     

Inhibitory effect of luteolin on osteoclast differentiation and function

Osteoclasts are multinucleated cells that play a crucial role in bone resorption, and are formed by the fusion of mononuclear osteoclasts derived from osteoclast precursors of the macrophage lineage. Compounds that specifically target functional osteoclasts would be ideal candidates for anti-resorptive agents for clinical applications. In the present study, we investigated the effects of luteolin, a flavonoid, on the regulation of receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis, functions and signaling pathway. Addition of luteolin to a coculture system of mouse bone marrow cells and ST2 cells in the presence of 10⁻⁸ M 1α,25(OH)₂D₃ caused significant inhibition of osteoclastogenesis. Luteolin had no effects on the 1α,25(OH)₂D₃-induced expressions of RANKL, osteoprotegerin and macrophage colony-stimulating factor mRNAs. Next, we examined the direct effects of luteolin on osteoclast precursors using bone marrow macrophages and RAW264.7 cells. Luteolin completely inhibited RANKL-induced osteoclast formation. Moreover, luteolin inhibited the bone resorption by mature osteoclasts accompanied by the disruption of their actin rings, and these effects were reversely induced by the disruption of the actin rings in mature osteoclasts. Finally, we found that luteolin inhibited RANKL-induced osteoclastogenesis through the suppression of ATF2, downstream of p38 MAPK and nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) expression, respectively. Taken together, the present results indicate that naturally occurring luteolin has inhibitory activities toward both osteoclast differentiation and functions through inhibition of RANKL-induced signaling pathway as well as actin ring disruption, respectively.     

SNX10 is required for osteoclast formation and resorption activity

RANKL-stimulation of osteoclast precursors results in up-regulation of genes involved in the process of differentiation and activation. In this report we describe the expression and functional characterization of Sorting Nexin 10 (snx10). Snx10 belongs to the sorting nexin (SNX) family, a diverse group of proteins with a common feature: the PX domain, which is involved in membrane trafficking and cargo sorting in endosomes. Snx10 is strongly up-regulated during RANKL-induced osteoclast differentiation in vitro and expressed in osteoclasts in vivo. qPCR analysis confirmed a significant increase in the expression of snx10 in in vitro-derived osteoclasts, as well as in femur and calvaria. Immunohistochemical analysis of mouse embryo sections showed expression in long bone, calvariae, and developing teeth. The expression was limited to cells that also expressed TRAP, demonstrating osteoclastic localization. Confocal immunofluorescence and subcellular fractionation analysis revealed Snx10 localization in the nucleus and in the endoplasmic reticulum (ER). To study a possible role for snx10 in osteoclast differentiation and function we silenced snx10 expression and found that snx10 silencing inhibited RANKL-induced osteoclast formation and osteoclast resorption on hydroxyapatite. Silencing also inhibited TRAP secretion. Taken together, these results confirm that snx10 is expressed in osteoclasts and is required for osteoclast differentiation and activity in vitro. Since inhibition of vesicular trafficking is essential for osteoclast formation and activity and SNX10 is involved in intracellular vesicular trafficking, these studies may identify a new candidate gene involved in the development of human bone diseases including osteoporosis.     

c-Src control of chloride channel support for osteoclast HCl transport and bone resorption

Bone degradation by osteoclasts depends upon active transport of hydrogen ions to solubilize bone mineral. This transport is supported by the parallel actions of a proton ATPase and a chloride channel located in the osteoclast ruffled membrane. We have previously identified a novel chloride channel, p62, which appears to be the avian counterpart to CLIC-5b and is expressed coincident with the appearance of acid secretion as avian osteoclasts differentiate in culture. In this article, we show that suppression of CLIC-5b in differentiating avian osteoclasts results in decreased acidification by vesicles derived from these cells and decreased ability of the cells to resorb bone. Acidification is rescued by the presence of valinomycin, consistent with a selective loss of chloride channel but not proton pump activity. Osteoclast bone resorption is known to be dependent on the expression of the tyrosine kinase, c-Src. We show that CLIC-5b from osteoclasts has affinity for both Src SH2 and SH3 domains. We find that suppression of expression of Src in developing osteoclasts results in decreased vesicular acidification, which is rescued by valinomycin, consistent with the loss of chloride conductance in the proton pump-containing vesicles. Suppression of c-Src causes no change in the steady state level of CLIC-5b expression, but does result in failure of proton pump and CLIC-5b to colocalize in cultured osteoclast precursors. We conclude that suppression of c-Src interferes with osteoclast bone resorption by disrupting functional co-localization of proton pump and CLIC-5b.