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1 Division of Biochemistry, Department of Molecular and Cellular Biology, Kobe University Graduate School of Medicine, Kobe, Japan
2 Division of Gastroenterological Surgery, Department of Surgery, Kobe University Graduate School of Medicine, Kobe, Japan
3 Department of Medical Oncology, The First Affiliated Hospital, China Medical University, Shenyang, China
4 Division of Biochemistry, Faculty of Pharmaceutical Sciences, Himeji Dokkyo University, Himeji, Japan
5 Department of Pharmaceutical Health Care, Faculty of Pharmaceutical Sciences, Himeji Dokkyo University, Himeji, Japan
6 Child Health and Care Section, Department of Health Care Sciences, Himeji Dokkyo University, Himeji, Japan
7 Department of Laboratory Medicine, Kawasaki Medical School, Kurashiki, Japan
8 Hyogo Prefectural Institute of Public Health and Environmental Sciences, Kobe, Japan
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Abstract |
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-tubulin is essential for the whole process of osteolysis under the control of a tyrosine kinase Syk. By developing a traceable and reproducible in vitro analyzing system for osteoclast function, we found that ATP-signaling gives rise to two events simultaneously (i) cytoskeletal reorganization for the formation of sealing zones, ring-like adhesion structures which delimit the contact surface, and (ii) the delivery and secretion of lytic granules towards the delimited site on the matrix. We further found that deacetylation of -tubulin is a critical reaction for osteoclast function. Pharmacological inhibition of -tubulin deacetylation resulted in (i) failure of the sealing-zone like structure formation and (ii) ceased secretion of lytic granules. Additionally, kinetics of deacetylation was found to be regulated by Syk. These data suggest a novel P2X7 microtubular regulation pathway related to Syk for a therapeutic target in osteolytic diseases. |
Introduction |
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Osteoclasts are multinucleated cells that are known to be generated as a result of fusion of mononuclear macrophages, and degrade the bone by releasing the acidic granules containing degradative enzymes into the delimited space on the bone matrix (Takayanagi 2007; Teitelbaum 2007).
More specifically, osteoclasts develop specialized actin-rich adhesion structures, so-called podosomes, which subsequently undergo dramatic reorganization into sealing zones. These ring-like adhesion structures, which delimit the resorption site, effectively seal the cell to the substrate forming a diffusion barrier. The structural integrity of the sealing zone is essential for the cell ability to degrade the bone, yet its structural organization is poorly understood. In addition, what molecule acts as the stimulating factor of the sealing-zone formation and how the structural change is linked with the release of acidic granules towards the bone matrix are still unknown.
In recent years, increasing attention has been paid to extracellular ATP signaling which specifically induces release of an inflammatory cytokine IL-1β in macrophages by the P2X7 receptor (Carta et al. 2006; Ferrari et al. 2006; Qu et al. 2007; Pelegrin et al. 2008). All cell types express cell surface receptors for extracellular nucleotides named P2 receptors that are divided into two types of receptors; the G-protein-coupled P2Y receptors and the ionotropic P2X receptors (Khakh & North 2006). Osteoclasts express multiple P2 receptors (Naemsch et al. 1999; Hoebertz et al. 2003) including P2X7 and P2X7 receptor has been shown to play an important role in the regulation of bone formation and/or resorption (Chiozzi et al. 1997; Morrison et al. 1998; Jorgensen et al. 2002; Gartland et al. 2003; Ke et al. 2003; Korcok et al. 2004).
In the osteoclast function, essential requirement of several protein tyrosine kinases is reported, such as c-Src, Pyk2, Syk and c-fms. Especially about Syk, the roles of osteoclast differentiation are reported (Mócsai et al. 2004; Faccio et al. 2005; Shinohara et al. 2008), and furthermore it is noteworthy that osteoclast function through the integrin vβ3 activation (Zou et al. 2007) and the M-CSF-DAP12-pathway has been studied (Zou et al. 2008). Integration of the clues about the complex but important roles of Syk is indispensable for total understanding of osteoclast function.
In this study, we show that an energy molecule ATP acts as a specific osteolysis initiator via P2X7 nucleotide receptor, which gives rise to the sealing-zone formation and the secretion of lytic granules. By the use of pharmacological inhibitor of deacetylase, we also show that deacetylation of -tubulin is essential for the above two events. Finally, we show that the regulation of -tubulin deacetylation is under the control of Syk.
These results suggest a P2X7-microtubular regulation pathway related to Syk is a therapeutic target in osteolytic diseases.
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Results |
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To develop an in vitro study of osteolysis, CD14-positive monocytes were isolated from human peripheral blood and differentiated under the osteoclast-inducing condition, plated on vitronectin-coated (a ligand of integrin vβ3) glass-dish or dentin slices (Zou et al. 2007; Sørensen et al. 2007). After three weeks in culture, multinucleated, TRAP-positive and cathepsinK-positive mature osteoclasts were obtained (Fig. 1A,B). Approximately 1.5 x 103 mature osteoclasts were obtained from 1 x 105 CD14-positive monocytes.
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For extracellular ATP signaling, there are two types of cell-surface receptors: the G-protein-coupled P2Y receptors and the ionotropic P2X receptors. As for osteoclasts, previous studies have demonstrated the presence of multiple subtypes of P2Y and P2X receptors, including P2Y1, P2Y2, P2X2, P2X4 and P2X7 on the cell surface. Bz-ATP is a more potent agonist than ATP at P2X7, it is also known to activate a number of P2 receptors in addition to P2X7. To identify the receptor, by which the effect of ATP on pit formation is mediated, we tested the effects of agonists at other P2 receptors. Osteoclasts were stimulated with low concentration of ATP (10 µM), which activates P2X4 and P2Y2 receptors, or high concentration of ATP (3 mM), which activates P2X7 receptors specifically for a short duration (20 min). As a result, the stimulation with high concentration of ATP for a short duration increased the number of resorption pits but low concentration of ATP did not (Fig. 1D). In addition, osteoclasts were stimulated with 150 µM UTP (P2Y2 agonist) or 100 µM ADP (P2Y1 agonist), but neither of the two P2Y agonists showed the pit formation (Fig. 1D). Pre-treatment with Coomassie brilliant blue G (BBG), a selective P2X7 antagonist (Jiang et al. 2000; Armstrong et al. 2009) completely inhibited Bz-ATP-induced pit formation (Fig. 1D). These results indicate that extracellular ATP acts as an initiating factor of osteolysis and P2X7 is a potent candidate receptor on osteoclasts. Furthermore, we confirmed that P2X7 receptors are expressed on the cell surface of osteoclasts derived from monocytes, and ATP stimulation promotes the traffic of the cell surface P2X7 receptors to lysosomes (Fig. 1E).
ATP-signaling induces the formation of sealing-zone like structure via the reorganization of cytoskeleton
We next investigated how ATP induces the reorganization of cytoskeleton for sealing-zone formation in osteoclasts. Before ATP stimulation, osteoclasts formed a variety of cell shapes and most cells contained dotted actin-rich adhesive structure named podosomes, a special structure characterized in monocyte-macrophage lineage cells (Kopp et al. 2006; Chabadel et al. 2007; Destaing et al. 2008) and some cells already showed a ring-shaped distribution of podosomes similar to the sealing zone, which is a hallmark of bone-resorbing osteoclasts, as previously described (Gil-Henn et al. 2007; Luxenburg et al. 2007) (ATP (–) in Fig. 2A). The stimulation with ATP promptly and completely destroyed the preexistent actin cytoskeletal structure (5 min in Fig. 2A). Subsequently, dotted podosomes were newly formed and the distribution gradually changed and a few cells showed sealing-zone like structure (60 min in Fig. 2A). To examine the process of the sealing-zone formation, we followed the time course of the change of actin structure and classified the cells into five stages by the distribution of podosomes at the adherent site (Fig. 2B). To be specific, we defined the cells whose actin structure was destroyed just after ATP treatment as Stage1 (Fig. 2B). The cells were defined as Stage2 whose dotted podosomes were newly formed and the distribution was gradually changed to fill a round area (Fig. 2B). Next, at the center of the round area, a few podosomes were detached, and a small hole appeared (Stage3 in Fig. 2B). With the enlargement of the central hole, podosomes revealed a doughnut-shaped distribution similar to the sealing-zone built by osteoclasts on the bone surface and formed the isolated space under the plasma membrane inside the structure (Stage4 in Fig. 2B). The width of the doughnut-shaped sealing-zone like structure became thin with time and the cells became flatly attached (Stage5 in Fig. 2B). Figure 2C shows the proportion of the cells at the individual Stages in time course study and indicates that the sealing zone is formed in stages, according to the reorganization of actin cytoskeleton. Next, to identify the receptor by which ATP-induced sealing-zone formation is mediated, we examined the effects of agonists at various P2 receptors as shown in the analysis of resorption pits. Osteoclasts were stimulated with high concentration of ATP (3 mM) for a short duration (20 min) which specifically activates P2X7 receptor, low concentration of ATP (10 µM) which activates both P2X4 and P2Y2 receptors, 100 µM ADP (P2Y1 agonist), or 150 µM UTP (P2Y2 agonist). As shown in Fig. 2D, the stimulation with high concentration of ATP completely destroyed preexistent actin structure (0.5 h) and subsequently sealing zone was formed (2 h), but the stimulation with other agonists led to little change in actin cytoskeleton (0.5 h). These results suggest that ATP-induced sealing-zone formation is mainly mediated by P2X7. To further clarify the involvement of P2X7 on sealing-zone formation, we examined the effects of shRNA against P2X7 by using lentivirus vector system. As shown in Fig. 2E, sealing zone was unformed in osteoclasts whose expression of P2X7 was suppressed by treatment with shRNA against P2X7 but the similar effect was not observed in osteoclasts treated by control shRNA, indicating the specific involvement of P2X7 receptor.
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-tubulin, which is an essential component of the MTOC, was also redistributed (Fig. 3B). In this way, ATP stimulation led to the dynamic change of microtubule structure and resulted in reversible scattering of MTOC in accordance with the reorganization of actin structure in stages.
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Secretion of lysosmal granules and the formation of sealing-zone like structure are cooperatively performed
Next, to confirm whether ATP stimulation leads to secretion of acidic granules containing degaradative enzymes (designated as osteolytic granules), the movement of cathepsinK was chased by the time-lapse observation of the same cells. After ATP stimulation, the granules containing cathepsinK were gradually delivered towards the contact site and released into the culture medium (Fig. 4A). Then the relation between the delivery of osteolytic granules and the formation of sealing-zone like structure was analyzed in individual Stages after ATP stimulation.
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These data indicate that both the secretion of osteolytic granules and the formation of sealing zone are cooperatively performed (Fig. 4B,C). The series of granule delivery was also confirmed by the movie of the living cells (Supporting Information/Supplementary Material S1). Schematic diagram of this process is shown in Fig. 4E.
The above results led us to the question of which molecular events regulate and coordinate the sealing-zone formation and the delivery of osteolytic granules. We examined the degree of -tubulin acetylation at the lysine (K40) residue in osteoclasts after ATP stimulation, because -tubulin is acetylated reversibly and the degree of acetylation is known to contribute to the cell shape stabilization and the regulation of vesicular transport (Dompierre et al. 2007; Tran et al. 2007).
As shown in Fig. 5A, before ATP stimulation acetylated -tubulin distributed diffusely like dashed lines in the cytoplasm. Just after ATP stimulation acetylated -tubulin accumulated near the MTOC, and next the degree of acetylation was dramatically enhanced (Stage2), and was gradually decreased. Finally, the acetylation was restricted only inside the crater-like structure (Stage4). Because -tubulin-acetylation coincides with podosome formation and subsequently deacetylation occurred in accordance with the progress of sealing-zone like structure, we suggested that deacetylation after the increased acetylation of -tubulin is required for the osteoclast function. As histone deacetylase 6 (HDAC6) is known to deacetylate -tubulin (Hubbert et al. 2002; Serrador et al. 2004) and a few of the histone deacetylase inhibitors, TSA and SAHA have a beneficial effect on -tubulin deacetylation (Koeller et al. 2003; Cabrero et al. 2006), we used these inhibitors in our ATP-induced osteolytic system. With TSA or SAHA pre-treatment, -tubulin was hyperacetylated (Fig. 5B and data not shown) and both ATP-dependent increase of resorption pits (Fig. 5C and data not shown) and release of cathepsinK into the culture medium (Fig. 5D) were completely inhibited. These results indicate that -tubulin deacetylation is indispensable for the secretion of osteolytic granules.
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These results demonstrated that the change of acetylation/deacetylation status of -tubulin is essential for ATP-induced formation of the sealing-zone like structure, delivery and secretion of osteolytic granules from osteoclasts.
Syk plays an indispensable role for the osteoclast function by regulating -tubulin deacetylation
Next we addressed the question what kinds of molecules regulate this acetylation/deacetylation status of -tubulin. Previous studies implicated the GTP-binding protein RhoA in the control of -tubulin acetylation mediated by HDAC6 in osteoclasts (Destaing et al. 2005) and we recently reported that a tyrosine kinase Syk acts at the upstream of RhoA signaling in phagocytosis (Shi et al. 2006). Therefore, we examined whether Syk contributes to osteolysis by affecting acetylation/deacetylation status of -tubulin.
To determine whether Syk is involved in ATP-induced osteolysis, tyrosine-phosphorylation of Syk in the presence or absence of ATP was analyzed in monocyte-derived osteoclasts. Syk was tyrosine-phosphorylated before ATP treatment on vitronectin-coated dish and the phosphorylation was maintained for 2 h and declined (data not shown). Then to furthermore investigate the effect of Syk we used human leukemic HL60 cells. Both HDAC6 and Syk were equally expressed in HL60 cells and CD14-positive monocyte-derived osteoclasts (Fig. 6A upper left). HL60 cells were differentiated into macrophages with the pre-treatment with vitamin D3 and 12-O-tetradecanoylphorbol-13-acetate (TPA), subsequently cultured in the presence of sRANKL for 7 days and became multinucleated (Fig. 6B right) and TRAP-positive (data not shown) osteoclast-like cells. In these cells on vitronectin-coated dish, Syk was originally tyrosine-phosphorylated before ATP treatment and its phosphorylation was not increased (data not shown). Then, to confirm the involvement of Syk in ATP/P2X7 signaling, flag-tagged Syk was transiently transferred into HL60 cells by lentiviral system. As shown in Fig. 6A (lower left), tyrosine-phosphorylation of Syk was increased after the stimulation with ATP (0.5 mM 1 h). Next, to examine the relation between Syk and deacetylase, the association of Syk with HDAC6, a candidate of deacetylase, was analyzed. As shown in Fig. 6A (right), HDAC6 was coprecipitated with Syk in an ATP-dependent fashion.
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We examined the lytic activity of dentin slices in these cells after ATP treatment. The cell-surface expression of P2X7 receptors was identical between parental and mutant cells by flow cytometry (data not shown). As shown in Fig. 6C, parental HL60-derived osteoclast-like cells showed a marked increase in the number of resorption pits in an ATP-dependent fashion, but shRNA-Syk/HL cells showed little increase and rescue of Syk restored this increase. These results indicate that Syk is indispensable for lytic activity of dentin slices in osteoclast-like differentiated cells. Next, we examined whether Syk is concerned with -tubulin deacetylation. To examine the kinetics of deacetylation, HL60 and mutant HL60 cells were pretreated with TSA for 3 h. In this pre-treatment, -tubulin was equally hyperacetylated among these cells. After removal of TSA, the kinetics of deacetylation of -tubulin was quantitatively analyzed in a time-course study. Notably, the rate of deacetylation was reduced in shRNA-Syk/HL cells but restored by rescue of Syk (Fig. 6D). These results suggest that Syk regulates activity of deacetylase such as HDAC6.
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Discussion |
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B is activated in osteoclasts (Korcok et al. 2004). Recently it has been reported that activation of P2X7 specifically induces Ca2+ dependent translocation of PKC
to the basolateral membrane domain of osteoclasts (Armstrong et al. 2009). Consequently, these signaling might contribute to regulation of bone resorption. However, P2X7 gene-deficient mice showed deficient periosteal-bone formation, whereas longitudinal bone growth was not altered (Ke et al. 2003). Probably because the P2X7 receptor plays a role in intercellular communication between osteoblasts and osteoclasts as described previously (Jorgensen et al. 2002), and are connected with bone metabolism and bone mineralization (Panupinthu et al. 2008). Deletion of the P2X7 might work effectively on both osteoclasts and osteoblasts equally. As for the source of extracellular ATP, lysosome is a potential target. It is likely that ATP released from lytic granules acts as extracellular ligands, and ligated nucleotide receptors shuttle between lysosomes and cell surface in accord with the recent reports on other systems (Qureshi et al. 2007; Zhang et al. 2007). In fact, ATP stimulation promoted the traffic of the cell surface P2X7 receptors to lysosomes (Fig. 1E) and the fluorescence-labeled ATP was incorporated into lysosomes as well as P2X7 receptors (data not shown). In bone marrow microenvironment, ATP might be rather secreted by neighboring cells such as osteoblasts.
Next, we found that ATP-signaling induces the formation of sealing zone; a specialized structure for bone resorption through the coordinated reorganization of both actin and microtubules in osteoclasts. And the secretion of osteolytic granules is also cooperatively performed with the cytoskeletal changes. It is interesting to note that ATP-signaling destroyed preexistent actin structure totally and built the sealing zone from the beginning. However, the structure of microtubule changed in the unique way, that is, MTOC became scattered and formed a rim of crater-like structure. Recently, one report showed that MTOCs are structurally diverse and highly dynamic (Lüders & Stearns 2007). Another report showed that centrosome polarization delivers lytic granules to the immunological synapse (Stinchcombe et al. 2006). Taken together, this reversible change of MTOC might be important in the release of osteolytic granules.
To clarify a candidate signaling for the sealing-zone formation, we analyzed the effects of Ca2+ influx. As shown in Fig. 3C, Ca2+ influx is not required for ATP-induced rapid destruction of preexisting actin structure but required for the subsequent podosome formation, suggesting that influx of Ca2+ through the ionic channel P2X receptor is indispensable for sealing-zone completion. In addition to the previous studies (Jorgensen et al. 2002; Armstrong et al. 2009), Ca2+ signaling through activation of P2X7 might act at the upstream signaling of the sealing-zone formation in osteoclasts.
We furthermore show that deacetylation of -tubulin is a critical reaction for bone resorption by osteoclasts. Pharmacological inhibition of -tubulin deacetylation resulted in defective bone resorption, accompanied by (i) failure of sealing-zone formation and (ii) ceased secretion of osteolytic granules. A candidate deacetylase HDAC6 was reported to combine with microtubules via the dynein motor protein and play an important role in lysosomal transport (Kawaguchi et al. 2003), and therefore a similar mechanism might function in osteoclasts. In addition, we found that Syk regulates the deacetylation of -tubulin.
As for Syk in osteoclasts, the roles in both differentiation (Mócsai et al. 2004; Faccio et al. 2005; Shinohara et al. 2008) and its function to resorb bone have been reported. Especially about the function, the roles of Syk in the integrin vβ3 activation signaling and in DAP12-coupled c-Fms-signaling have been intensively studied (Zou et al. 2007, 2008) and these reports showed the importance of Syk-mediated organization of the osteoclast cytoskeleton through the interaction between ITAM proteins and SH2 domain of Syk. In addition to these data, we have demonstrated a novel function of Syk in bone-resorbing process, that is, the regulation of deacetylation status of microtubules at the upstream of deacetylation. Recently, we reported that Syk acts at the upstream of RhoA signaling in integrin Mβ2-dependent phagocytosis (Shi et al. 2006) and previous studies implicated the RhoA in the control of -tubulin acetylation in osteoclasts (Destaing et al. 2005).
Together with our current data, ATP/P2X7 signaling might act as inside-out signaling of integrin vβ3, which recruits Syk bound to the tyrosine-phosphorylated ITAM proteins in an SH2 domain-dependent manner. Furthermore, activated Syk regulates a deacetylase such as HDAC6 via Vav-RhoA pathway, and this novel pathway links the reorganization of cytoskeleton and the delivery of lytic granules. In fact, Syk was preactivated in osteoclasts and osteoclast-like differentiated cells on vitronectin-coated plates probably via integrin vβ3 signaling and ATP-dependent Syk activation was detected in wild-type Syk-transferred osteoclast-like differentiated HL60 cells (Fig. 6A). In differentiated condition, the signaling mediated by other P2 receptors including P2X and P2Y receptors and the signaling by integrin vβ3 might crosstalk each other. Decreased phosphorylation of Syk 2 h after ATP treatment might be related to partial detachment of the cells for ring-like structure formation.
As a controversial point, we obtained the data that defect of Syk also suppresses the ratio of -tubulin acetylation in in vitro analysis (data not shown). We propose the possibility that there may exist a common adaptor protein that has the ability to bind to both acetyltransferase and deacetylase and that Syk may regulate to which enzyme the adaptor protein is susceptible to binding. The role of Syk in the process of -tubulin acetylation remains to be solved.
Osteoclasts are of immunohematological origin and involved in crosstalk between the immune and bone systems (Takayanagi 2007). In fact, it is reported that cathepsinK, an osteoclast-specific enzyme, plays a critical role in TLR9 signaling in the immune system (Asagiri et al. 2008), and semaphorin-signaling is reported to be required not only in immune response but also in bone homeostasis (Takegahara et al. 2006). The present data show that -tubulin deacetylation is a critical reaction of bone resorption and suggest that the histone deacetylase inhibitors may act as potential molecular-targeting agents for osteolytic diseases. Originally, histone deacetylase inhibitors were developed as novel clinical agents in cancer therapy and may have side effects on bone metabolism, but this treatment potentially generates additional benefit on bone marrow cancer metastasis. Our data also may contribute to the proposal of Syk inhibitors as therapeutic drugs on osteoporosis and rheumatoid arthritis.
In this study, we show that an energy molecule ATP acts as a specific osteolysis initiator via P2X7 nucleotide receptor, which gives rise to the sealing-zone formation and the secretion of lytic granules. We also show that deacetylation of -tubulin is essential for the above two events under the control of Syk. From these results, we propose the importance of ATP/P2X7-microtubule deacetylation pathway related to Syk as potential therapeutic targets in osteolytic diseases.
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Experimental procedures |
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Reagents and antibodies
Rabbit antihuman Syk polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used for immunoblotting and immunoprecipitation. Rabbit antihuman HDAC6 polyclonal antibody (Santa Cruz Biotechnology) was used for immunoblotting. Agarose-conjugated anti-phosphotyrosine monoAb (clone 4G10) was used for immunoprecipitation (Millipore, Bedford, MA, USA). Mouse antihuman -tubulin and antihuman acetylated -tubulin, mouse antihuman -tubulin antibodies (Sigma, St Louis, MO, USA) were used to detect the microtubule distribution, modification and MTOC-formation, and Alexa Fluor594- and 488-conjugated phalloidin (Invitrogen, Carlsbad, CA, USA) were used to detect the actin-cytoskeleton. Confocal images were acquired by using a confocal laser-scanning microscope (LSM 510 META; Carl Zeiss, Oberkochen, Germany). Osteolytic granules were labeled with LysoTracker Red (Invitrogen) and Magic Red cathepsinK detection kit (Immunochemistry Technologies LLC, Bloomington, MN, USA). CathepsinK detection kit was also used to quantify the released enzyme activity in the culture medium in the presence or absence of ATP (Sigma). ADP, 2'-3'-O-(4-benzoyl-benzoyl)-ATP (Bz-ATP), UTP and Coomassie brilliant blue G were purchased (Sigma) and used to confirm the specific receptor of ATP which promotes osteolytic signaling, and Trichostatin A (TSA, Sigma) and Suberoylanilide hydroxamic acid (SAHA, Alexis Biochemicals, Lausen, Switzerland) were used for pre-treatment before the ATP-stimulation to examine the effects of deacetylation. Bodipy FL-conjugated ATP (Invitrogen) was used to chase the movement of ATP and rabbit antihuman P2X7 antibody (Millipore) was used to chase the movement of the ATP-bound receptor and for flow cytometry.
Separation of human primary monocytes, cell culture and differentiation into osteoclasts
The study dealing with the blood from healthy volunteers had been approved by the Ethical Committee of Himeji Dokkyo University, and the samples were handled after informed consent. Human monocytes were collected using the magnetic cell sorting (MACS) system and microbeads conjugated with monoclonal mouse antihuman CD14 antibody purchased-irom Miltenyi Biotec (Bergisch Gladbach, Germany). Briefly, peripheral blood mononuclear cells of healthy blood donors were isolated by density-gradient centrifugation using Ficoll-Paque (Pharmacia Biotech AB, Uppsala, Sweden). CD14-expressing cells were positively separated by MACS according to the protocol provided by the manufacturer.
The isolated cells were plated onto 10 µg/mL vitronectin-coated slide (BD Biosciences, San Jose, CA, USA) or dentin slices (Primary Cell Co. Ltd., Ishikari, Japan) and cultured in the presence of 10–7 M Vitamin D3 (Calbiochem, San Diego, CA, USA), 25 ng/mL M-CSF (R&D Systems, Minneapolis, MN, USA) and 10 ng/mL sRANKL (Peprotech EC, London, UK, added at six days after starting the culture) for ca. three weeks. Osteoclast differentiation was morphologically confirmed by multi-nuclei formation (Hoechst33342 and May-Gruenwald-Giemsa staining) and tartrate-resistant acid phosphatase (TRAP) staining (Sigma). HL60 cells were differentiated into osteoclasts on 10 µg/mL vitronectin-coated slide or dentin slices and in the medium containing 25 ng/mL M-CSF, 10–7 M Vitamin D3 and 10 ng/mL 12-O-tetradecanoylphorbol-13-acetate (TPA, Sigma) for two days, and furthermore cultured in the presence of 10 ng/mL sRANKL for five days. Osteoclast differentiation was confirmed as above described.
In vitro bone resorption assay
CD14-positive monocytes and HL60 cells differentiated into osteoclasts on dentin slices were used. Such treated cells were preincubated in the presence or absence of HDAC inhibitors for 3 h and next stimulated with 0.5 mM ATP or 300 µM Bz-ATP, for 20 h. In some experiments, osteoclasts derived from CD14-positive monocytes were stimulated with 100 µM ADP, 150 µM UTP, 10 µM ATP, for 20 h or with 3 mM ATP for 20 min and then cultured for 20 h, or stimulated with 300 µM Bz-ATP in the presence or absence of 10 µM BBG on dentin slices. The resorption pits on the slices were visualized after removing the cells (Chloroform:methanol = 2 : 1 solution) and washing with phosphate-buffered saline (PBS). The pits were stained with Carrazi's hematoxylin for 30 min, washed with PBS and observed by an inverted microscope.
Visualization of the cytoskeleton and lysosomal movement
The cells were differentiated into osteoclasts for three weeks and the cell images were visualized before and after using a confocal laser-scanning microscope. Serial z-sections were taken at 1.1 µM interval of individual cells. LSM 510 META software was used to generate x–z or y–z– planes as single image composites of serial x–y sections and to show the projections of the serial x–y images on the sealing zone that exists at the contact site.
Staging of ATP-induced sealing-zone formation and correlation with the osteolytic granules
The confocal images of actin cytoskeleton of osteoclasts stained by Alexa Fluor 488-conjugated phalloidin after ATP stimulation were divided by the distribution of podosomes. Stage1: the actin cytoskeletal structure is completely destroyed; Stage2: dotted podosomes newly formed are distributed to fill a round area; Stage3: a small hole (smaller than half radius of the cell size) appears in the center of the round podosome area; Stage4: podosomes reveal a doughnut-shaped distribution (the hole is larger than half radius and smaller than four-fifth of the cell size); Stage5: the sealing zone becomes thin (the hole is larger than four-fifth of the cell size). In some cases, to examine the effects of Ca2+ influx, The osteoclasts were preincubated and stimulated with ATP in Ca2+-free PBS or Ca2+-containing PBS (0.9 mM CaCl2).
Distribution of osteolytic granules correlated with the progression of sealing-zone formation is classified by projected serial confocal sections of lysosomes into three patterns: (i) before podosome formation; (ii) lysosomal distribution larger than the hole inside the sealing zone; (iii) lysosomal distribution smaller than the hole inside the sealing zone.
Time-lapse imaging of (1) ATP-dependent centripetal delivery of osteolytic granules and (2) detection of cathepsinK
The cells differentiated into osteoclasts for three weeks were used for time-laps imaging. (1) Living cells pretreated with LysoTracker Red were plated onto a culture dish, placed on the stage of fluorescence microscopy (Olympus IX81; Olympus, Tokyo, Japan) at 37 °C in 5% CO2. After preincubation for 30 min, time-lapse images were acquired at 15 s intervals and analyzed with AquaCosmos 2.6 software (Hamamatsu Photonics K.K., Hamamatsu, Japan).
Time-lapse movies were created at 15 fps using QuickTime Pro (Apple, Cupertino, CA). From the series of time-lapse photos, the direction of lysosomal movement was classified into three patterns: (i) towards the cell center; (ii) nondirectional or paused; (iii) towards the cell margin. At each condition, 14 vesicles per cell (n = 8) were calculated. (2) Living cells pretreated with cathepsinK substrates which showed the fluorescence depending on the enzymatic activity were plated onto a culture dish and placed on the stage of a confocal laser-scanning microscopy (LSM 510 META) at 37 °C in 5% CO2. After preincubation for 30 min, time-lapse images were acquired at 10 min intervals and analyzed.
Quantification of the rate of (1) acetylation and (2) deacetylation of -tubulin
(1) The cells were pretreated with 10 µM nocodazole for 1.5 h, washed three times with PBS and then incubated under the culture condition for the indicated times. Cells were fixed with 4% paraformaldehyde, stained with anti-acetylated -tubulin antibody and the length of the acetylated part of microtubules that newly extended from the MTOC was measured and the average length was calculated on each cell. (2) The cells were pretreated with 1 µM TSA for 3 h, washed three times with PBS and then incubated under the culture condition for the indicated times. Cells were fixed, stained with anti-acetylated -tubulin antibody and the ratio of fluorescence intensity was analyzed with AquaCosmos 2.6 software.
Plasmids and transfection
To inhibit syk gene expression, oligonucleotides for the sense and antisense target sequences of the human syk-coding region including bp 1192–1210 with stem-loop sequence were synthesized and constructed into the pSilencer 2.1-U6 hygro (Ambion, Austin, TX). To support the knockdown effect of Syk-shRNA, Flag-rescue-Syk expression vectors (pcDNA4-TO) containing four silent mismatches in the knockdown oligonucleotide sequence were constructed as previously described (Shi et al. 2006). These plasmids were introduced into HL60 cells by electroporation and positive clones were selected with hygromycin (Wako Pure Chemicals Industries, Osaka, Japan) or zeocin (Invitrogen). Furthermore, to confirm the involvement of Syk in ATP/P2X7-signaling Flag-human Syk cDNA expression vector (pLenti6/V5-D-TOPO) was constructed and introduced into HL60 cells by lentiviral expression system (Invitrogen, Carlsbad, CA, USA), and successful transfection was confirmed by immunoblotting analysis. To inhibit P2X7 gene expression, P2X7-shRNA/pLKO.1-puro and shRNA nontarget control/pLKO.1-puro (Sigma) were purchased and introduced into oseteoclasts derived from CD14-positive monocytes by lentiviral expression system (Invitrogen).
Immunoprecipitation and immunoblotting
Cells were lysed with lysis buffer (0.05% SDS, 0.5% sodium deoxycholate, 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 2 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM Na3VO4). Cellular debris was sonicated and removed by centrifugation. In some cases, cell lysate was immunoprecipitated with anti-Syk antibody at 4 °C for 60 min, and then incubated with Protein G-Sepharose beads for 60 min, washed three times and subjected to immunoblotting. For detection of tyrosine phosphorylation proteins, agarose-conjugated anti-phosphotyrosine monoAb (clone 4G10) was directly used. Cell lysates or immunoprecipitated samples were separated in SDS–PAGE and transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was blocked with 5% skim milk in T-TBS (25 mM Tris–HCl (pH 8.0), 150 mM NaCl, 0.1% Tween 20) for 60 min at room temperature and then incubated with the appropriate antibodies. The membrane was washed three times with T-TBS and incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibodies for 30 min, and specific proteins were detected using an enhanced chemiluminescence immunoblotting system.
Statistical analysis
In some experiments, statistical significance was determined by the Student's t-test.
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Acknowledgements |
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This research was supported by KAKENHI (18590267 and 20590321); a Grant-in-Aid for Scientific Research (C) from Japan Society for the Promotion of Sciences and the Osaka Medical Research Foundation for Incurable Diseases.
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Footnotes |
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* Correspondence: ytohyama{at}himeji-du.ac.jp
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References |
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Received: 15 November 2008
Accepted: 27 April 2009
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