Effects of odanacatib on bone-turnover markers in osteoporotic postmenopausal women: a post hoc analysis of the LOFT study.

This post hoc analysis and modeling study examined the mechanism of action of odanacatib using a statistical model to explain sCTx response in ODN-treated patients as a function of other bone-turnover biomarkers that, with other observed biomarker changes, showed that odanacatib persistently inhibited osteoclastic bone removal activity without preventing osteoclastogenesis. INTRODUCTION: Odanacatib (ODN) is an oral selective cathepsin K (CatK) inhibitor, previously in development for osteoporosis treatment. A post hoc analysis examined ODN's mechanism of action on bone-turnover biomarkers. METHODS: A subset of patients who completed 60 months' treatment in the Long-Term Odanacatib Fracture Trial (LOFT; NCT00529373) (N = 112 [57 ODN, 55 placebo]) were evaluated. Serum (s) and urine (u) samples were assayed at baseline and months 6-60 for 10 known bone-remodeling biomarkers: sCTx, uαα- and ußßCTx/Cr, uNTx/Cr, sNTx, uDPD/Cr, sICTP, sTRAP5b, sPINP, and sBSAP. Because the CrossLaps® CTx assay identifies the CTx peptide as well as larger molecular weight CTx-containing peptides, including ICTP, a best-fit model was developed to explain the transient sCTx reduction in ODN-treated patients. RESULTS: ODN persistently reduced the bone-resorption markers sNTx, uNTx/Cr, uαα- and ußßCTx/Cr, and uDPD/Cr, and gradually increased the target-engagement marker sICTP and osteoclast number (sTRAP5b), versus placebo from baseline to month 60. sCTx was transiently reduced with ODN within 12 months, returning to baseline by month 48. Modeling suggested that sCTx changes in the ODN group were primarily due to increased accumulation of larger CTx species, including sICTP. The bone-formation markers sPINP and sBSAP showed partial reductions, versus placebo, in the first 6 months but approached baseline by months 48-60. CONCLUSION: Observed changes in bone-turnover biomarkers support the persistent efficacy of ODN in direct inhibition of osteoclastic bone-resorption activity, without inhibition of osteoclastogenesis. Long-term evaluation also underscores the unique mechanism of ODN on osteoclastic collagen processing and subsequently osteoblastic bone formation. TRIAL REGISTRATION: NCT00529373.

Primary clear cell sarcoma of the tongue and surgical reconstruction: About a rare case report.

Clear cell sarcomas (SCC), also called "soft-tissue melanoma", are rare and aggressive tumors that preferentially affect the lower limbs (tendons and fasciae) and which have also been described in head and neck localizations. Their clinical and immunohistochemical mimicry with melanoma makes it difficult to diagnose sarcomas. SCC treatment is mainly focused on large-scale resection surgery with adjuvant radiotherapy because of their low chemo-sensitivity and extreme lymphophilia. In case of head and neck localization, these treatments may lead to function and aesthetic sequelae thus requiring the use of modern techniques of reconstructive surgery. The authors describe the diagnosis, treatment and follow-up of large lingual SCC case using a DIEP free flap reconstruction according to an original technique developed in the department. Given the characteristics of patients with SCC (a high proportion of women between 20 and 40 years old) and its inherent qualities (low morbidity of the donor site, volume delivered and excellent plasticity), the fascio-cutaneous free flap type "DIEP" "taken according to the design of the" Cathedral triptych seems to be a viable choice among the range of reconstruction solutions.

Effects of long term treatment with high doses of odanacatib on bone mass, bone strength, and remodeling/modeling in newly ovariectomized monkeys.

The objectives here were to evaluate the effects of odanacatib (ODN) at doses exceeding the clinical exposure on biomechanical properties of lumbar vertebrae (LV), hip and central femur (CF), and compare ODN to alendronate (ALN) on bone remodeling/modeling in ovariectomized (OVX) monkeys. Ten days post-surgery, animals were treated with vehicle (VEH), ODN-L (2mg/kg/day, p.o.), ODN-H (8/4mg/kg/day), or ALN (30µg/kg/week, s.c.) for 20months. An intact group was also included. ODN-L provided systemic exposures of 1.8-fold of clinical exposure. ODN-H started at 20-fold for 5.5months, and then reduced to 7.8-fold of clinical exposure, compared to ALN at approximated clinical exposure. From cross sectional analyses, LV density and peak load in ODN at both doses or ALN were not different from VEH or Intact. However, cortical thickness of femoral neck (FN) and CF in ODN were higher (21-34%, p<0.05) than VEH, due to smaller endocortical (Ec) perimeter of FN (10-11%; p<0.05) and CF (9-12%; ODN-L, p<0.05), and larger CF periosteal (Ps) perimeter (2-12%; ODN-H, p<0.001) versus VEH. ODN groups also showed slightly higher cortical porosity and Ps non-lamellar bone in CF. ODN-H treatment resulted in higher CF peak load (p<0.05) versus VEH. For all bone sites analyzed, a positive, linear relationship (r(2)=0.46-0.69, p<0.0001) of peak load to density or structural parameters was demonstrated. No treatment-related differences in the derived intrinsic strength properties were evidenced as compared between groups. ALN reduced all remodeling surfaces without affecting Ps modeling. Trabecular and intracortical remodeling were reduced in ODN groups, similar to ALN. Ec mineralizing surface in ODN-H trended to be lower than VEH by month 20, but Ec bone formation indices in ODN groups generally were not different from VEH. Ps modeling in ODN groups was significantly higher than other treatment groups. This study overall demonstrated the bone safety profile of ODN and its unique mechanism on cortical bone supporting the clinical application for osteoporosis treatment.

Inhibition of bone resorption by the cathepsin K inhibitor odanacatib is fully reversible.

The cathepsin K (CatK) inhibitor odanacatib (ODN) is currently being developed for the treatment of osteoporosis. In clinical trials, efficacy and resolution of effect of ODN treatment on bone turnover biomarkers and accrued bone mass have been demonstrated. Here, we examine the effects of continuing treatment and discontinuation of ODN versus alendronate (ALN) on osteoclast (OC) function. First, accessibility and reversible engagement of active CatK in intracellular vesicles and resorption lacunae of actively resorbing OCs were demonstrated by the selective and reversible CatK inhibitors, BODIPY-L-226 (IC50=39nM) and L-873,724 (IC50=0.5nM). Next, mature human OCs on bone slices were treated with vehicle, ODN, or ALN for 2days, followed by either continuing with the same treatment, or replacement of the inhibitors by vehicle for additional times as specified per experimental conditions. Maintaining OCs on ODN or ALN significantly reduced CTx-I release compared to vehicle controls. However, only the treatment of OCs with ODN resulted in the formation of small shallow discrete resorption pits, retention of intracellular vesicles enriched with CatK and other lysosomal enzymes, increase in 1-CTP release and number of TRAP(+) OCs. Upon discontinuation of ODN treatment, OCs rapidly resumed bone resorption activity, as demonstrated by a return of OC functional markers (CTx-I, 1-CTP), cell number and size, morphology and number of resorption pits, and vesicular secretion of CatK toward the respective vehicle levels. As expected, discontinuation of ALN did not reverse the treatment-related inhibition of OC activity in the time frame of the experiment. In summary, this study demonstrated rapid kinetics of inhibition and reversibility of the effects of ODN on OC bone resorption, that differentiated the cellular mechanism of CatK inhibition from that of the bisphosphate antiresorptive ALN.

The effects of the cathepsin K inhibitor odanacatib on osteoclastic bone resorption and vesicular trafficking.

Odanacatib (ODN) is a selective, potent and reversible inhibitor of cathepsin K (CatK) that inhibits bone loss in postmenopausal osteoporosis. Evidence from osteoclast (OC) formation from bone marrow of CatK(-/-) mice or human OC progenitors treated with ODN, demonstrated that CatK inhibition has no effect on osteoclastogenesis or survival of OCs. Although having no impact on OC activation, ODN reduces resorption activity as measured by CTx release (IC(50)=9.4 nM) or resorption area (IC(50)=6.5 nM). While untreated cells generate deep trail-like resorption lacunae, treated OCs form small discrete shallow pits. ODN leads to significant accumulation of intracellular vesicles intensely stained for CatK and TRAP. CatK (+) vesicles localize toward the basolateral and functional secretory membranes of the polarized OC and TRAP(+) vesicles evenly distribute in the cytoplasm, suggesting that ODN disrupts multiple vesicular trafficking pathways. Intracellular levels of both precursor and mature TRAP were increased by 2-fold and the pre-pro and mature CatK by 6- and 2-fold in ODN-treated OCs compared to untreated controls. ODN treated OC accumulates labeled degraded bone matrix proteins in CatK containing vesicles. In summary, ODN treatment inhibits bone resorption by blocking degradation of demineralized collagen in the resorption lacunae, and retarding transcytosis for further processing of degraded proteins.

A new osteophyte segmentation algorithm using partial shape model and its applications to rabbit femur anterior cruciate ligament transection via micro-CT imaging.

Osteophyte is an additional bony growth on a normal bone surface limiting or stopping motion at a deteriorating joint. Detection and quantification of osteophytes from CT images is helpful in assessing disease status as well as treatment and surgery planning. However, it is difficult to distinguish between osteophytes and healthy bones using simple thresholding or edge/texture features due to the similarity of their material composition. In this paper, we present a new method primarily based active shape model (ASM) to solve this problem and evaluate its application to anterior cruciate ligament transection (ACLT) rabbit femur model via CT imaging. The common idea behind most ASM based segmentation methods is to first build a parametric shape model from a training dataset and apply the model to find a shape instance in a target image. A common challenge with such approaches is that a diseased bone shape is significantly altered at regions with osteophyte deposition misguiding an ASM method and eventually leading to suboptimum segmentations. This difficulty is overcome using a new partial ASM method that uses bone shape over healthy regions and extrapolates it over the diseased region according to the underlying shape model. Finally, osteophytes are segmented by subtracting partial-ASM derived shape from the overall diseased shape. Also, a new semi-automatic method is presented in this paper for efficiently building a 3D shape model for an anatomic region using manual reference of a few anatomically defined fiducial landmarks that are highly reproducible on individuals. Accuracy of the method has been examined on simulated phantoms while reproducibility and sensitivity have been evaluated on CT images of 2-, 4- and 8-week post-ACLT and sham-treated rabbit femurs. Experimental results have shown that the method is highly accurate ( R2 = 0.99), reproducible (ICC = 0.97), and sensitive in detecting disease progression (p-values: 0.065,0.001 and < 0.001 for 2- vs. 4, 4- vs. 8- and 2- vs. 8-weeks, respectively).

Effect of the cathepsin K inhibitor odanacatib on bone resorption biomarkers in healthy postmenopausal women: two double-blind, randomized, placebo-controlled phase I studies.

Inhibition of cathepsin K (CatK) is a potential new treatment for osteoporosis. In two double-blind, randomized, placebo-controlled phase I studies, postmenopausal female subjects received odanacatib (ODN), an orally active, potent, and selective CatK inhibitor, once weekly for 3 weeks or once daily for 21 days. Bone turnover biomarkers, safety monitoring, and plasma ODN concentrations were assessed. These studies showed ODN to be well tolerated. Pharmacokinetic (PK) analysis revealed a long half-life (t(1/2); 66-93 h) consistent with once-weekly dosing. Pronounced reductions in C-terminal telopeptide of type I collagen (approximately 62%) and N-terminal telopeptide of type I collagen normalized to creatinine (NTx/Cr) (approximately 62%) at trough (C(168 h)) were seen following weekly administration. Robust reductions in CTx (up to 81%) and NTx/Cr (up to 81%) were seen following daily administration. ODN exhibits robust and sustained suppression of bone resorption biomarkers (CTx and NTx/Cr) at weekly doses > or = 25 mg and daily doses > or = 2.5 mg.

Non-peptide alpha(v)beta(3) antagonists: identification of potent, chain-shortened RGD mimetics that incorporate a central pyrrolidinone constraint.

Antagonists of the integrin receptor alpha(v)beta(3) are expected to have utility in the treatment of osteoporosis through inhibition of bone resorption. A series of potent, chain-shortened, pyrrolidinone-containing alpha(v)beta(3) receptor antagonists is described. Two sets of diasteromeric pairs of high-affinity antagonists demonstrated marked differences in log P values, which translated into differing dog pharmacokinetic properties. One member of this set was demonstrated to be effective in reducing bone resorption in rats.

Abnormal localisation and hyperclustering of (alpha)(V)(beta)(3) integrins and associated proteins in Src-deficient or tyrphostin A9-treated osteoclasts.

The non-receptor tyrosine kinase Src was shown to be essential for osteoclast function in vivo. We have previously reported that engagement of (alpha)(v)(beta)(3) integrin in osteoclasts induces tyrosine phosphorylation and activation of the adhesion kinase PYK2 and the adaptor protein p130(Cas) in a Src-dependent manner. The objective of this study was to analyse the role of c-Src in the (alpha)(v)(beta)(3) integrin-dependent recruitment of signalling and cytoskeletal molecules in osteoclasts during bone resorption. Using prefusion osteoclasts (pOCs) obtained from cocultures of osteoblasts and spleen cells isolated from Src(-/-) mice or their normal littermates, we found: (1) similar expression levels and ligand binding affinities of (alpha)(v)(beta)(3) integrins in Src(-/-) and Src(+/?) pOCs, (2) reduced adhesion and spreading of Src(-/-) pOCs, (3) defective organisation of the microfilament proteins, F-actin, vinculin and paxillin, and of PYK2 and p130(Cas) in the sealing zone of Src(-/-)OCLs, and (4) hyperclustering of (alpha)(v)(beta)(3) integrins together with microfilament and signalling proteins in the basal membrane of Src-deficient OCLs. In normal OCLs, the tyrosine kinase inhibitor tyrphostin A9 inhibits actin ring formation, bone resorption and tyrosine phosphorylation of several proteins, including c-Src. Furthermore, tyrphostin A9 induced similar hyperclustering of (alpha)(v)(beta)(3) integrins in osteoclasts as observed in Src(-/-) OCLs. Taken together, these findings suggest that normal localisation of (alpha)(v)(beta)(3) and recruitment of its downstream effectors to the appropriate compartments of the osteoclast during resorption depend on Src kinase activity.

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.

PYK2 is an adhesion kinase in macrophages, localized in podosomes and activated by beta(2)-integrin ligation.

Pyk2 is a member of the focal adhesion kinase (FAK) family, highly expressed in the central nervous system and haemopoietic cells. Although Pyk2 is homologous to FAK, its role in signaling pathways was shown to be distinct from that of FAK. We show here that Pyk2 is highly expressed in peritoneal IC-21 macrophage and is tyrosine phosphorylated in response to cell attachment to fibronectin and fibrinogen. Upon IC-21 cell adhesion, Pyk2 tyrosine phosphorylation is inhibited by blocking antibodies to the integrin subunits alpha(M) and beta(2). Furthermore, Pyk2 is rapidly tyrosine phosphorylated in response to ligation of beta(2) integrins by antibodies. In migrating macrophages, Pyk2 localizes to perinuclear regions and to podosomes, where it is clustered with tyrosine phosphorylated proteins. Furthermore, in the podosomal ring structure, which surrounds the central actin core, Pyk2 co-localizes with vinculin, talin, and paxillin. In the podosomes, Pyk2 also co-localizes with the integrin alpha(M)beta(2). Lastly, reduction of Pyk2 expression in macrophages leads to inhibition of cell migration. We propose that Pyk2 is functionally linked to the formation of podosomes where it mediates the integrin-cytoskeleton interface and regulates cell spreading and migration.

Nonpeptide alpha(v)beta(3) antagonists. 1. Transformation of a potent, integrin-selective alpha(IIb)beta(3) antagonist into a potent alpha(v)beta(3) antagonist.

Modification of the potent fibrinogen receptor (alpha(IIb)beta(3)) antagonist 1 generated compounds with high affinity for the vitronectin receptor alpha(v)beta(3). Sequential modification of the basic N-terminus of 1 led to the identification of the 5,6,7, 8-tetrahydro[1,8]naphthyridine moiety (THN) as a lipophilic, moderately basic N-terminus that provides molecules with excellent potency and selectivity for the integrin receptor alpha(v)beta(3). The THN-containing analogue 5 is a potent inhibitor of bone resorption in vitro and in vivo. In addition, the identification of a novel, nonpeptide radioligand with high affinity to alpha(v)beta(3) is also reported.

Integrins and signaling in osteoclast function.

Integrins are heterodimeric adhesion receptors that mediate cell-matrix and cell-cell interactions. Osteoclasts highly express the alphavbeta3 integrin, which binds to a variety of extracellular matrix proteins including vitronectin, osteopontin and bone sialoprotein. RGD-containing peptides, RGD-mimetics and alphavbeta3 blocking antibodies inhibit bone resorption in vitro and in vivo, suggesting that this integrin plays an important role in osteoclast function. RGD-containing peptides were shown to raise cytosolic calcium in osteoclasts. Furthermore, several signaling and adaptor molecules were found to be involved in alphavbeta3 integrin-dependent signaling pathways, including phosphatidylinositol 3-kinase, c-Src, PYK2 and p130(cas). In addition, cytoskeletal molecules such as paxillin, vinculin, gelsolin and F-actin are recruited to adhesion contacts upon integrin activation. Many of these molecules signaling and cytoskeletal localize to the sealing zone of actively resorbing osteoclasts, suggesting that they play a role in linking the adhesion of osteoclasts to the bone matrix with the cytoskeletal organization and the polarization and activation of these cells for bone resorption.

Role of alpha(v)beta(3) integrin in osteoclast migration and formation of the sealing zone.

The alpha(v)beta(3) integrin is abundantly expressed in osteoclasts and has been implicated in the regulation of osteoclast function, especially in cell attachment. However, in vivo studies have shown that echistatin, an RGD-containing disintegrin which binds to alpha(v)beta(3), inhibits bone resorption without changing the number of osteoclasts on the bone surface, suggesting inhibition of osteoclast activity. The objective of this study was to examine how occupancy of alpha(v)beta(3) integrins inhibits osteoclast function, using primary rat osteoclasts and murine pre-fusion osteoclast-like cells formed in a co-culture system. We show that: (1) echistatin inhibits bone resorption in vitro at lower concentrations (IC(50 )= 0.1 nM) than those required to detach osteoclasts from bone (IC(50 ) approximately 1 microM); (2) echistatin (IC(50 )= 0.1 nM) inhibits M-CSF-induced migration and cell spreading of osteoclasts; (3) alpha(v)beta(3) integrins are localized in podosomes at the leading edge of migrating osteoclasts, whereas, with echistatin treatment (0.1 nM), alpha(v)beta(3) disperses randomly throughout the adhesion surface; and (4) when bone resorption is fully inhibited with echistatin, there is visible disruption of the sealing zone (IC(50 )= 13 nM), and alpha(v)beta(3) visualized with confocal microscopy re-distributes from the basolateral membranes to intracellular vesicular structures. Taken together, these findings suggest that alpha(v)beta(3) integrin plays a role in the regulation of two processes required for effective osteoclastic bone resorption: cell migration (IC(50 )= 0.1 nM) and maintenance of the sealing zone (IC(50) approximately 10 nM).

Stable association of PYK2 and p130(Cas) in osteoclasts and their co-localization in the sealing zone.

Bone resorption is initiated by osteoclast attachment to the mineralized matrix, cytoskeletal reorganization, cellular polarization, and the formation of the sealing zone. The present study examines the interaction between PYK2 and p130(Cas) (Crk-associated substrate), suggested to be part of the signaling pathway initiated by osteoclast adhesion. Using murine osteoclast-like cells (OCLs) and their mononuclear precursors (pOCs), generated in a co-culture of bone marrow and osteoblastic MB1.8 cells, we show that: 1) p130(Cas) is tyrosine-phosphorylated upon adhesion of pOCs to vitronectin or ligation of beta3 integrins; 2) p130(Cas) colocalizes with PYK2 and the cytoskeletal proteins F-actin, vinculin, and paxillin in the podosomal-rich ring-like structures of OCLs plated on glass and in the sealing zone in actively resorbing OCLs on bone; 3) p130(Cas) and PYK2 form a stable complex in pOCs, independent of tyrosine phosphorylation of either molecule, and this complex is present in Src (-/-) OCLs, in which neither protein is phosphorylated or associated with the osteoclast adhesion structure; 4) the association of p130(Cas) and PYK2 is mediated by the SH3 domain of p130(Cas) and the C-terminal domain of PYK2. These findings suggest that p130(Cas) and its association with PYK2 may play an important role in the adhesion-dependent signaling that leads to cytoskeletal reorganization and formation of the sealing zone during osteoclast activation.

Interleukin 1 induces multinucleation and bone-resorbing activity of osteoclasts in the absence of osteoblasts/stromal cells.

Interleukin-1 (IL-1) is one of the most potent bone-resorbing factors involved in bone loss associated with inflammation. We previously reported that IL-1 prolonged the survival of multinucleated osteoclast-like cells (OCLs) formed in cocultures of murine osteoblasts/stromal cells and bone marrow cells via the prevention of spontaneously occurring apoptosis. It was reported that macrophage colony-stimulating factor (M-CSF/CSF-1) prolongs the survival of OCLs without the help of osteoblasts/stromal cells. The present study was conducted to determine whether IL-1 also directly induces the multinucleation and activation of OCLs. Mononuclear osteoclast-like cells (prefusion osteoclasts; pOCs) were purified using the "disintegrin" echistatin from cocultures of murine osteoblastic cells (MB 1.8 cells) and bone marrow cells. Both IL-1 and M-CSF prolonged the survival and induced the multinucleation of pOCs through their respective receptors. However, actin ring formation (a functional marker of osteoclasts) by multinucleated cells was observed in the pOC cultures treated with IL-1, but not those treated with M-CSF. We previously reported that enriched multinucleated OCLs as well as pOCs placed on bone/dentine slices formed few resorption pits, but their pit-forming activity was greatly increased by the addition of osteoblasts/stromal cells. Here, pit-forming activity of both pOCs and enriched OCLs placed on dentine slices was induced by adding IL-1, even in the absence of osteoblasts/stromal cells. M-CSF failed to induce pit-forming activity in pOC and enriched OCL cultures. These results indicate that IL-1 induces the multinucleation and bone-resorbing activity of osteoclasts even in the absence of osteoblasts/stromal cells.

Echistatin inhibits the migration of murine prefusion osteoclasts and the formation of multinucleated osteoclast-like cells.

The vitronectin receptor alpha(v)beta3 is highly expressed in osteoclasts and was shown to play a critical role in osteoclast function in vivo. The objective of this study was to examine the role of alpha(v)beta3 integrin in osteoclast formation in vitro using the inhibitory disintegrin echistatin, an RGD-containing snake venom. We documented by immunocytochemistry and Northern blot analysis that during murine osteoclast-like cell (OCL) formation in a coculture of mouse osteoblastic MB1.8 cells and bone marrow cells there is increased expression of the alpha(v) and beta3 integrin subunits. Echistatin binds preferentially to the membrane fraction of isolated enriched OCLs (IC50 = 0.6 nM), and this binding is inhibited by vitronectin receptor-blocking polyclonal antibodies. Additionally, cross-linking of radiolabeled echistatin to OCLs, followed by immunoprecipitation with antibodies to vitronectin or fibronectin receptors, shows that alpha(v)beta3 integrin is the predominant receptor for echistatin in this system. In this coculture, echistatin completely inhibits the formation of multinucleated OCLs, but not that of mononuclear prefusion OCLs (pOCs). This inhibition is RGD and dose dependent (IC50 = 0.7 nM). We tested the hypothesis that inhibition of OCL formation may be due to interference with pOC migration and found that echistatin inhibited macrophage colony-stimulating factor-induced migration and fusion of pOCs (IC50 = 1 and 0.6 nM, respectively). Echistatin inhibition of pOCs migration and fusion is also RGD dependent. These results suggest that the integrin alpha(v)beta3 plays a role in pOC migration, which can explain the inhibitory effect of echistatin on multinucleated osteoclast formation in vitro.

PYK2 in osteoclasts is an adhesion kinase, localized in the sealing zone, activated by ligation of alpha(v)beta3 integrin, and phosphorylated by src kinase.

Osteoclast activation is initiated by adhesion to the bone surface, followed by cytoskeletal rearrangement, the formation of the sealing zone, and a polarized ruffled membrane. This study shows that PYK2/CAKbeta/RAFTK, a cytoplasmic kinase related to the focal adhesion kinase, is highly expressed in rat osteoclasts in vivo. Using murine osteoclast-like cells (OCLs) or their mononuclear precursors (pOCs), generated in a coculture of bone marrow and osteoblastic MB1.8 cells, we show: (a) tyrosine phosphorylation of PYK2 upon ligation of beta3 integrins or adhesion of pOCs to serum, vitronectin, osteopontin, or fibronectin but not to laminin or collagen; (b) coimmunoprecipitation of PYK2 and c-Src from OCLs; (c) PYK2 binding to the SH2 domains of Src; (d) marked reduction in tyrosine phosphorylation and kinase activity of PYK2 in OCLs derived from Src (-/-) mice, which do not form actin rings and do not resorb bone; (e) PYK2 phosphorylation by exogeneous c-Src; (f) translocation of PYK2 to the Triton X-100 insoluble cytoskeletal fraction upon adhesion; (g) localization of PYK2 in podosomes and the ring-like structures in OCLs plated on glass and in the sealing zone in OCLs plated on bone; and (h) activation of PYK2, in the presence of MB1.8 cells, parallels the formation of sealing zones and pit resorption in vitro and is reduced by echistatin or calcitonin and cytochalasin D. Taken together, these findings suggest that Src-dependent tyrosine phosphorylation of PYK2 is involved in the adhesion-induced formation of the sealing zone, required for osteoclastic bone resorption.