Actin Ring Formation In Osteoclasts Research Articles

Flavonoids from the peels of Citrus unshiu Markov. and their inhibitory effects on RANKL-induced osteoclastogenesis through the downregulation of c-Fos signaling in vitro

Phytochemical investigation of Citrus unshiu peels led to the isolation of eight new flavonols (7–9, 11–15) and sixteen known compounds (1–6, 10, 16–24). Their structures were elucidated using spectroscopic analysis (1D, 2D NMR, and HR-MS). Besides, all isolated compounds (1–24) were evaluated for their inhibitory effects on receptor activator of RANKL-induced osteoclastogenesis in BMMs. Among them, dimethylmikanin (1), quercetogetin (2), 3,3′,4′,5,7,8-hexamethoxyflavone (3), 3-methoxynobiletin (4) showed a significant inhibitory effect on RANKL-induced osteoclast differentiation at a concentration of 10 μM. Moreover, 3-methoxynobiletin (4) suppressed RANKL-induced osteoclastogenesis by decreasing the number of osteoclasts and osteoclast actin-ring formation in a dose-dependent manner without causing any cytotoxic effects on BMMs. At the molecular level, 3-methoxynobiletin (4) inhibited RANKL-induced c-Fos expression and subsequently NFATc1 activation, as well as the expression of osteoclastogenesis-related marker genes c-Src and CtsK. These findings suggested that 3-methoxynobiletin (4) attenuated osteoclast differentiation by inhibiting RANKL-mediated c-Fos signaling and that it may have therapeutic potential for treating or preventing bone resorption-related diseases, such as osteoporosis.

A novel regulatory role of TRAPPC9 in L-plastin-mediated osteoclast actin ring formation.

Trafficking protein particle complex 9 (TRAPPC9) is a major subunit of the TRAPPII complex. TRAPPC9 has been reported to bind nuclear factor κB kinase subunit β (IKKβ) and NF-kB-inducing kinase (NIK) where it plays a role in the canonical and noncanonical of nuclear factor-κB (NF-kB) signaling pathways, receptively. The role of TRAPPC9 in protein trafficking and cytoskeleton organization in osteoclast (OC) has not been studied yet. In this study, we examined the mRNA expression of TRAPPC9 during OC differentiation. Next, we examined the colocalization of TRAPPC9 with cathepsin-K, known to mediate OC resorption suggesting that TRAPPC9 mediates the trafficking pathway within OC. To identify TRAPPC9 protein partners important for OC-mediated cytoskeleton re-organization, we conducted immunoprecipitation of TRAPPC9 in mature OCs followed by mass spectrometry analysis. Our data showed that TRAPPC9 binds various protein partners. One protein with high recovery rate is L-plastin (LPL). LPL localizes at the podosomes and reported to play a crucial role in actin aggregation thereby actin ring formation and OC function. Although the role of LPL in OC-mediated bone resorption has not fully reported in detail. Here, first, we confirmed the binding of LPL to TRAPPC9 and, then, we investigated the potential regulatory role of TRAPPC9 in LPL-mediated OC cytoskeleton reorganization. We assessed the localization of TRAPPC9 and LPL in OC and found that TRAPPC9 is colocalized with LPL at the periphery of OC. Next, we determined the effect of TRAPPC9 overexpression on LPL recruitment to the actin ring using a viral system. Interestingly, our data showed that TRAPPC9 overexpression promotes the recruitment of LPL to the actin ring when compared with control cultures. In addition, we observed that TRAPPC9 overexpression reorganizes actin clusters/aggregates and regulates vinculin recruitment into the OC periphery to initiate podosome formation.

Microtubule actin crosslinking factor 1 (MACF1) knockdown inhibits RANKL-induced osteoclastogenesis via Akt/GSK3β/NFATc1 signalling pathway

Osteoclasts are responsible for bone resorption and play essential roles in causing bone diseases such as osteoporosis. Microtubule actin crosslinking factor 1 (MACF1) is a large spectraplakin protein that has been implicated in regulating cytoskeletal distribution, cell migration, cell survival and cell differentiation. However, whether MACF1 regulates the differentiation of osteoclasts has not been elucidated. In this study, we found that the expression of MACF1 was increased in primary bone marrow-derived monocytes (BMMs) of osteoporotic mice and was downregulated during receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis of pre-osteoclast cell lines RAW264.7 cells. RAW264.7 cells were transfected with shMACF1 using a lentiviral vector to study the role of MACF1 in osteoclastogenic differentiation. Knockdown of MACF1 in RAW264.7 cells inhibited the formation of multinucleated osteoclasts and decreased the expression of osteoclast-marker genes (Ctsk, Acp5, Mmp9 and Oscar) during RANKL-induced osteoclastogenesis. Additionally, knockdown of MACF1 disrupted actin ring formation in osteoclasts and further blocked the bone resorption activity of osteoclasts by reducing the area and depth of pits. Knockdown of MACF1 had no effect on the survival of pre-osteoclasts and mature osteoclasts. We further established that knockdown of MACF1 attenuated the phosphorylation of Akt and GSK3β and inhibited the expression of its downstream target NFATc1. Akt activator rescued the inhibition of osteoclast differentiation by MACF1 knockdown. These data demonstrate that MACF1 positively regulates osteoclast differentiation via the Akt/GSK3β/NFATc1 signalling pathway, suggesting that targeting MACF1 may be a novel therapeutic approach against osteoporosis.

Ethanol extract of Polyscias fruticosa leaves suppresses RANKL-mediated osteoclastogenesis in vitro and LPS-induced bone loss in vivo

BackgroundMany bone-related diseases such as osteoporosis and rheumatoid arthritis are commonly associated with the excessive activity of osteoclasts. Polyscias fruticosa has been used as traditional medicine for the treatment of ischemia and inflammation and also eaten as a salad. However, its effect on the bone related diseases has not been investigated yet. PurposeThis study aimed to investigate the effect of ethanol extract of P. fruticosa on RANKL-induced osteoclastogenesis in vitro and LPS-induced bone loss in mouse, and evaluate anti-osteoclastogenic activities of its major constituents. MethodsBMMs or RAW264.7 cells were treated with ethanol extract from P. fruticose leaves (EEPL), followed by an evaluation of cell viability, RANKL-induced osteoclast differentiation, actin-ring formation, and resorption pits activity. Effects of EEPL on RANKL-induced phosphorylation of MAPKs were evaluated by Western blotting. The expression levels of NFATc1 and c-Fos were evaluated by Western blotting or immunofluorescence assay. The expression levels of osteoclast-specific marker genes were evaluated by Western blotting and reverse transcription-qPCR analysis. A LPS-induced murine bone loss model was used to evaluate the protective effect of EEPL on inflammation-induced bone loss. HPLC analysis was performed to identify the major constituents of EEPL. ResultsEEPL significantly inhibited RANKL-induced osteoclast differentiation by decreasing the number of osteoclasts, osteoclast actin-ring formation, and bone resorption. EEPL suppressed RANKL-induced phosphorylation of p38 and JNK MAPKs, as well as the expression of c-Fos and NFATc1. EEPL decreased the expression levels of osteoclast marker genes, including MMP-9, TRAP and CtsK. Mice treated with EEPL significantly protected the mice from LPS-induced osteoclast formation and bone destruction as indicated by micro-CT and histological analysis of femurs. We also identified 3-O-[β-d-glucopyranosyl-(1→4)-β-d-glucuronopyranosyl] oleanolic acid 28-O-β-d-glucopyranosyl ester (1) and quercitrin (3) as the active constituents in EEPL for inhibiting RANKL-induced osteoclast differentiation. ConclusionThe results showed that EEPL exerted anti-osteoclastogenic activity in vitro and in vivo by inhibiting RANKL-induced osteoclast differentiation and function, and suggested that EEPL could have beneficial applications for preventing or inhibiting osteoclast-mediated bone diseases.

Ganomycin I from Ganoderma lucidum attenuates RANKL-mediated osteoclastogenesis by inhibiting MAPKs and NFATc1

Many bone-related diseases such as osteoporosis and rheumatoid arthritis are commonly associated with excessive activity of the osteoclast. Ganomycin I (GMI), a meroterpenoid isolated from Vietnamese mushroom Ganoderma lucidum, possesses a variety of beneficial effects on human health. However, its impact and underlying mechanism on osteoclastogenesis remain unclear. In the present study, we investigated the effect of GMI on RANKL-induced osteoclast formation in mouse BMMs and RAW264.7 cells. BMMs or RAW264.7 cells were treated with GMI followed by an evaluation of cell viability, RANKL-induced osteoclast differentiation, actin-ring formation, and resorption pits activity. Effects of GMI on RANKL-induced phosphorylation of MAPKs as well as the expression levels of NFATc1 and c-Fos were evaluated by Western blot analysis. Expression levels of osteoclast marker genes were evaluated by Western blot analysis and reverse transcription-qPCR. GMI significantly inhibited RANKL-induced osteoclast differentiation by decreasing the number of osteoclasts, osteoclast actin-ring formation, and bone resorption in a dose-dependent manner without affecting cell viability. At molecular level, GMI inhibited the RANKL-induced phosphorylation of ERK, JNK, and p38 MAPKs, as well as the expression levels of c-Fos and NFATc1, which are known to be crucial transcription factors for osteoclast formation. In addition, GMI decreased expression levels of osteoclastogenesis specific marker genes including c-Src, CtsK, TRAP, MMP-9, OSCAR, and DC-STAMP in RANKL-stimulated BMMs. Our findings suggest that GMI can attenuate osteoclast formation by suppressing RANKL-mediated MAPKs and NFATc1 signaling pathways and the anti-osteoclastogenic activity of GMI may extend our understanding of molecular mechanisms underlying biological activities and pharmacological use of G. lucidum as a traditional anti-osteoporotic medicine.

Sappanone A inhibits RANKL-induced osteoclastogenesis in BMMs and prevents inflammation-mediated bone loss

Receptor activator of nuclear factor-kB ligand (RANKL) is a key factor in the differentiation and activation of osteoclasts. Suppressing osteoclastogenesis is considered an effective therapeutic approach for bone-destructive diseases, such as osteoporosis and rheumatoid arthritis. Sappanone A (SPNA), a homoisoflavanone compound isolated from the heartwood of Caesalpinia sappan, has been reported to exert anti-inflammatory effects; however, the effects of SPNA on osteoclastogenesis have not been investigated. In the present study, we describe for the first time that SPNA inhibits RANKL-induced osteoclastogenesis in mouse bone marrow macrophages (BMMs) and suppresses inflammation-induced bone loss in a mouse model. SPNA inhibited the formation of osteoclasts from BMMs, osteoclast actin-ring formation, and bone resorption in a concentration-dependent manner. At the molecular level, SPNA significantly inhibited RANKL-induced activation of the AKT/glycogen synthase kinase-3β (GSK-3β) signaling pathway without affecting its activation of the mitogen-activated protein kinases (MAPKs) JNK, p38, and ERK. In addition, SPNA suppressed the induction of nuclear factor of activated T cells cytoplasmic 1 (NFATc1), which is a crucial transcription factor in osteoclast differentiation. As a result, SPNA decreased osteoclastogenesis-related marker gene expression, including CtsK, TRAP, dendritic cell-specific transmembrane protein (DC-STAMP), MMP-9 and osteoclast-associated receptor (OSCAR). In a mouse inflammatory bone loss model, SPNA significantly inhibited lipopolysaccharide (LPS)-induced bone loss by suppressing the number of osteoclasts. Taken together, these findings suggest that SPNA inhibits osteoclastogenesis and bone resorption by inhibiting the AKT/GSK-3β signaling pathway and may be a potential candidate compound for the prevention and/or treatment of inflammatory bone loss.

Regulation of osteoclast differentiation and actin ring formation by the cytolinker protein plectin

Osteoclasts are cells that resorb the bone matrix and maintain bone and calcium homeostasis. An actin ring is a characteristic actin structure that is essential for bone resorption by osteoclasts. Tyrosine kinase Src deficient osteoclasts do not form actin rings; thus, Src is a key molecule for actin ring formation in osteoclasts. However, how Src regulates actin ring formation is not fully understood. We identified the cytolinker protein plectin as a Src-binding protein by immunoprecipitation and liquid chromatography tandem mass spectrometry. Plectin is a huge protein (>500 kDa) and regulates the cytoskeleton by binding to actin and tubulin. We assessed the expression and role of plectin in osteoclasts. Plectin was expressed and co-localized with Src close to the actin ring in osteoclasts. Moreover, plectin was tyrosine-phosphorylated by Src. Differentiation and actin ring formation were inhibited by downregulation of plectin. These results suggest an important role for plectin in osteoclast differentiation and actin ring formation through Src binding.

Osteoclast-Primed Foxp3+ CD8 T Cells Induce T-bet, Eomesodermin, and IFN-γ To Regulate Bone Resorption.

Osteoimmunology arose from the recognition that cytokines produced by lymphocytes can affect bone homeostasis. We have previously shown that osteoclasts, cells that resorb bone, act as APCs. Cross-presentation of Ags by osteoclasts leads to expression of CD25 and Foxp3, markers of regulatory T cells in the CD8 T cells. Octeoclast-induced Foxp3(+) CD25(+) regulatory CD8 T cells (OC-iTcREG) suppress priming of CD4 and CD8 T cells by dendritic cells. OC-iTcREG also limit bone resorption by osteoclasts, forming a negative feedback loop. In this study, we show that OC-iTcREG express concurrently T-bet and Eomesodermin (Eomes) and IFN-γ. Pharmacological inhibition of IκK blocked IFN-γ, T-bet, and Eomes production by TcREG Furthermore, we show, using chromatin immunoprecipitation, NF-κB enrichment in the T-bet and Eomes promoters. We demonstrate that IFN-γ produced by TcREG is required for suppression of osteoclastogenesis and for degradation of TNFR-associated factor 6 in osteoclast precursors. The latter prevents signaling by receptor activator of NF-κB ligand needed for osteoclastogenesis. Knockout of IFN-γ rendered TcREG inefficient in preventing actin ring formation in osteoclasts, a process required for bone resorption. TcREG generated in vivo using IFN-γ(-/-) T cells had impaired ability to protect mice from bone resorption and bone loss in response to high-dose receptor activator of NF-κB ligand. The results of this study demonstrate a novel link between NF-κB signaling and induction of IFN-γ in TcREG and establish an important role for IFN-γ in TcREG-mediated protection from bone loss.

Inhibition of Osteoclastogenesis and Bone Resorption in vitro and in vivo by a prenylflavonoid xanthohumol from hops

Excessive RANKL signaling leads to superfluous osteoclast formation and bone resorption, is widespread in the pathologic bone loss and destruction. Therefore, targeting RANKL or its signaling pathway has been a promising and successful strategy for this osteoclast-related diseases. In this study, we examined the effects of xanthohumol (XN), an abundant prenylflavonoid from hops plant, on osteoclastogenesis, osteoclast resorption, and RANKL-induced signaling pathway using both in vitro and in vivo assay systems. In mouse and human, XN inhibited osteoclast differentiation and osteoclast formation at the early stage. Furthermore, XN inhibited osteoclast actin-ring formation and bone resorption in a dose-dependent manner. In ovariectomized-induced bone loss mouse model and RANKL-injection-induced bone resorption model, we found that administration of XN markedly inhibited bone loss and resorption by suppressing osteoclast activity. At the molecular level, XN disrupted the association of RANK and TRAF6, resulted in the inhibition of NF-κB and Ca2+/NFATc1 signaling pathway during osteoclastogenesis. As a results, XN suppressed the expression of osteoclastogenesis-related marker genes, including CtsK, Nfatc1, Trap, Ctr. Therefore, our data demonstrated that XN inhibits osteoclastogenesis and bone resorption through RANK/TRAF6 signaling pathways. XN could be a promising drug candidate in the treatment of osteoclast-related diseases such as postmenopausal osteoporosis.

The 1,2,3-triazole derivative KP-A021 suppresses osteoclast differentiation and function by inhibiting RANKL-mediated MEK-ERK signaling pathway.

The triazole family of compounds has been implicated in modulating various biological processes such as inflammation, tumorigenesis, and infection. To our knowledge, this is the first study to demonstrate the effects of 1,2,3-triazole substituted biarylacrylonitrile compounds, including KP-A021, on the differentiation and function of osteoclasts. KP-A021 and its triazole derivatives, at a concentration that does not cause a cytotoxic response in bone marrow macrophages (BMMs), significantly inhibited osteoclast differentiation induced by receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) as assessed by tartrate-resistant acid phosphatase (TRAP) staining. KP-A021 also dramatically inhibited the expression of marker genes associated with osteoclast differentiation, such as TRAP, cathepsin K (Cat K), dendritic cell-specific transmembrane protein (DC-STAMP), and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1). Furthermore, KP-A021 inhibited actin ring formation in osteoclasts as well as resorption pit formation induced by osteoclasts. Analysis of the signaling pathway for KP-A021 indicated that this triazole compound inhibited the RANKL-induced activation of extracellular signal-regulated kinase (ERK) and its upstream signaling molecule, mitogen-activated protein kinase kinase1/2 (MEK1/2). Taken together, these results demonstrate that KP-A021 has an inhibitory effect on the differentiation and function of osteoclasts via modulation of the RANKL-induced activation of the MEK-ERK pathway.

Inhibition of osteoclast actin ring formation and activation by phloretin through disturbing integrin αvβ3‐PYK2 pathway (1045.29)

Osteoclast activation is important for bone remodeling and is altered in multiple bone disorders such as osteoporosis. This process requires cell adhesion and extensive actin cytoskeletal reorganization. Phloretin, a dihydrochalcone and a type of natural phenols, can be found in apple tree leaves. The goal of this study was to define the process and mechanism of bone resorption. RAW 264.7 macrophages were incubated with 1‐20 μM phloretin for 5 days in the presence of RANKL. RANKL stimulated Akt activation and c‐Src expression, which was blunted by phloretin. The integrin αvβ3, heterodimer of adhesion receptors, is crucial in osteoclast activation mediating cell‐matrix and cell‐cell interactions. Phloretin suppressed integrin β3 induction and PYK2 activation elevated by RANKL, indicating its inhibition of osteoclastic fusion. The activation of PYK2, a major cell adhesion‐activated tyrosine kinase in osteoclasts, is required for the formation of actin rings. Taken together, these results suggest that phloretin was an inhibitor of actin ring formation, disrupting integrin‐PYK2 pathway for the regulation of actin cytoskeletal organization in osteoclasts.Grant Funding Source: Supported by Brain Korea 21 plus

Urocortin is a novel regulator of osteoclast differentiation and function through inhibition of a canonical transient receptor potential 1-like cation channel

This study investigated the role of urocortin (UCN), a member of the corticotrophin-releasing factor (CRF) family of peptides, in osteoclast maturation and function. We found that 10(-7) M UCN significantly (P<0.05) suppressed osteoclast differentiation from bone marrow precursor cells in culture and reduced the expression of several osteoclastic markers. Furthermore, UCN potently suppressed osteoclast bone resorption, by significantly inhibiting both the plan area of bone resorbed by osteoclasts and actin ring formation within osteoclasts at 10(-9) M (P<0.05), with complete inhibition at 10(-7) M (P<0.001). UCN also inhibited osteoclast motility (10(-7) M) but had no effect on osteoclast survival. Osteoclasts expressed mRNA encoding both UCN and the CRF receptor 2β subtype. Pre-osteoclasts however, expressed CRF receptor 2β alone. Unstimulated osteoclasts contained constitutively active cation channel currents with a unitary conductance of 3-4 pS, which were inhibited by over 70% with UCN (10(-7) M). Compounds that regulate calcium signalling and energy status of the cell, both crucial for osteoclast activity were investigated. The non-selective cation channel blockers, lanthanum (La(3)(+)) and gadolinium (Gd(3)(+)), inhibited actin ring formation in osteoclasts, whereas modulators of voltage-dependent Ca(2)(+) channels and K(ATP) channels had no effect. These findings show for the first time that UCN is a novel anti-resorptive molecule that acts through a direct effect on osteoclasts and their precursor cells.

Osteoclast actin ring formation occurs during in vitro culture with a diverse array of calcified substrates

Kurzbeschreibung: Einleitung: Die ausgepragten Beschwerden der Patienten und die damit verbundenen hohen Kosten fur das Gesundheitssystem nach traumatischen Knochenverletzungen, Osteoporose oder osteolytischen Knochenmetastasen, unterstreichen die Wichtigkeit der Entwicklung neuer Ansatze in der Knochenwiederherstellung. Bei groseren Knochenverlusten ist es notwendig Knochentransplantate beziehungsweise Knochenersatzmaterialien einzusetzen. Viele fuhren jedoch zu verschiedenen Komplikationen wie, unter anderem, Reaktionen des Immunsystems, Abstosung und auf diesem Weg ubertragbaren Infektionskrankheiten. Es ist notwendig, neue Strategien in der Knochenregeneration zu entwickeln. Um optimale Regeneration zu gewahrleisten, mussen die Eigenschaften der neuen Knochenersatzmaterialien vollstandig geklart sein. Ziel der Studie: Untersuchung der Faktoren, die in die Anhaftung an und die Reaktion von Osteoklasten (OC) auf mineralisierte Substrate beeinflussen, um die besten Bedingungen fur die fruhen Stadien der Knochenregeneration zu schaffen. Methoden: OC aus Kaninchen oder aus muriner Cokultur von OB mit OC-Vorstufen aus dem Knochenmark, wurden auf Knochen, Elfenbein, Koralle, Muschelschalen und Glas kultiviert. Die Organisation des Zytoskeletts wurde bei verschiedenen Temperaturbehandlungen der Substrate (auser Glas) bei 50°C, 200°C, 400° oder 600°C analysiert. Durch die Temperaturbehandlung wurden Modifikationen der Zusammensetzung der Substrate, wie Proteingehalt oder kristalliner Struktur, erreicht. Die Zellen wurden nach der Kultur auf den jeweiligen Substraten fixiert und das Aktin-haltige Zytoskelett wurde durch Rhodamin-Phalloidin mittels Fluoreszenzmikroskopie sichtbar gemacht. Resultat: Unabhangig von der Temperaturbehandlung, wurden keine schadigenden Effekte der Substrate (Aragonit, Calcit, Perlmutt, Knochen, Elfenbein) auf OC festgestellt. Interessanterweise, hafteten OC stark an Knochen, Elfenbein und den anderen kalzifizierten Substraten, unabhangig von geandertem Proteingehalt oder modifizierter kristalliner Struktur. OC entwickelten auf allen Substraten (ebenfalls unabhangig von der Temperaturbehandlung) Sealing zones, mit Ausnahme der Glasoberflachen, die als Kontrolle dienten. Bemerkenswerterweise wurden „podosomen belts“ auf allen Substraten, inklusive Knochen, entdeckt. Diese Entdeckung widerspricht fruheren Untersuchungen, nach denen „podosomen belts“ nicht auf Knochen erscheinen. Zusammengefasst zeigen die Ergebnisse, dass der Ausloseimpuls fur die Bildung von Sealing zones durch OC weder Apatit noch Kalziumphosphat ist. Kalziumkarbonat ist ausreichend um die Entstehung von Sealing zones zu ermoglichen. Schlussfolgerung: Die Substrate Aragonit, Calcit und Perlmutt sind in ahnlicher Weise wie Knochen von OC abbaubar, eine Erkenntnis die in die Entwicklung neuer Knochenersatzmaterialien einfliesen sollte. Offenlegung von Interessenskonflikten: Keine

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.

Inhibition of Osteoclast Function by Adenovirus Expressing Antisense Protein-tyrosine Kinase 2

Osteoclast activation is initiated by adhesion to bone, cytoskeletal rearrangement, formation of the sealing zone, and formation of the polarized ruffled membrane. Previous findings suggest that protein-tyrosine kinase 2 (PYK2), a cytoplasmic kinase related to focal adhesion kinase, participates in these events. This study examines the role of PYK2 in adhesion-mediated signaling and osteoclast function, using PYK2 antisense. We produced a recombinant adenovirus containing a 300-base pair reversed 5'-coding region of PYK2 and used full-length PYK2 as a control. Murine osteoclast-like cells or their mononuclear precursors were generated in a co-culture of bone marrow and osteoblasts. Infection with antisense adenovirus significantly reduced the expression of endogenous PYK2 protein relative to uninfected cells or to cells infected with sense PYK2 and caused: 1) a reduction in osteoclast formation in vitro; 2) inhibition of cell spreading and of actin ring formation in osteoclasts plated on glass or bone and of attachment and spreading of osteoclast precursors plated on vitronectin; 3) inhibition of bone resorption in vitro; 4) marked reduction in p130(Cas) tyrosine phosphorylation; and 5) no change in alpha(v)beta(3) integrin expression or c-Src tyrosine phosphorylation. Taken together, these findings support the hypothesis that PYK2 plays a central role in the adhesion-dependent cytoskeletal organization and sealing zone formation required for osteoclastic bone resorption.

Casein kinase 2 phosphorylation of recombinant rat osteopontin enhances adhesion of osteoclasts but not osteoblasts.

Osteopontin (OP) is a highly phosphorylated bone matrix protein and contains the RGD cell-binding motif, which mediates cell adhesion through integrin receptors that include alpha(v)beta3. Casein kinase 2 (CK2) is a factor-independent serine/threonine kinase, which may be the predominant physiologically relevant kinase for OP phosphorylation. This study was designed to examine the effects of unphosphorylated recombinant rat OP, and CK2-phosphorylated OP (P-OP), on the adhesion and function of mouse osteoclasts (OC) and osteoblast-like cells (UMR 201-10B and UMR 106-06) in vitro. OP significantly increased OC adhesion compared to plastic alone, and cell attachment was further increased at least twofold on OP phosphorylated with CK2. Attachment was dependent on the integrity of the RGD domain and was completely abolished in the presence of 1 mM RGD peptide. Neither CK2 phosphorylation of mutant OP, in which the RGD was converted to RGE or RAD, nor protein kinase C (PKC) phosphorylation of wild-type OP enhanced OC attachment. An antibody to the beta3 integrin subunit, but not anti-mouse CD44 antibody, specifically blocked the proportion of attachment due to phosphorylation of OP. Actin ring formation in OC was increased by plating cells onto OP, with no further increase by phosphorylation. Both OP and CK2-phosphorylated OP enhanced attachment of the two osteoblastic cell lines, compared to plastic, but in contrast to OCs, there was no significant difference with phosphorylation. Osteoblast attachment was totally blocked by 1 mM RGD peptide, but was not influenced by the beta3 integrin antibody. Plating of UMR 201-10B cells onto OP further increased retinoic acid-induced alkaline phosphatase expression. The results suggest that specific phosphorylation of OP is important for interaction with OCs, compared with osteoblastic cells, and that alternative integrins may be important in the interaction between osteoblastic cells and OP compared with OCs.

Regulation of osteoclast function.

Regulation of osteoclast function.

Chemical and physical properties of the extracellular matrix are required for the actin ring formation in osteoclasts.

To examine the effect of extracellular matrix on osteoclast polarization, we focused on the actin organization in osteoclasts, using murine osteoclast-like multinucleated cells (OCLs) formed in cocultures of osteoblastic cells and bone marrow cells. When OCLs were cultured on either a plastic plate, calcified dentine, or calcium phosphate thin films in the presence of fetal bovine serum (FBS), they similarly formed ringed structures of F-actin dots (actin rings). However, OCLs placed on demineralized dentine or type I collagen gel matrix (collagen gel) failed to form actin rings. In the absence of FBS, actin ring formation in OCLs was induced on plastic plates coated with vitronectin, fibronectin, or type I collagen, but not on those coated with laminin, poly-L-lysine, or bovine serum albumin. Actin ring formation appeared to depend on integrins, since the GRGDS, but not the GRGES, peptide inhibited it in a dose-dependent manner. Moreover, immunoelectron microscopic examination revealed that vacuolar proton ATPase (V-ATPase) was localized along the apical membrane in much higher densities than the basolateral membrane in OCLs placed on plastic coverslips. In OCLs placed on collagen gel, however, V-ATPase was found to be distributed throughout the cytoplasm without polarity. These results suggest that actin ring formation in osteoclasts was dependent on matrix substrates, matrix proteins and integrins, and was closely related to osteoclast function.