Specific Antagonists of NMDA Receptors Prevent Osteoclast Sealing Zone Formation Required for Bone Resorption

Abstract A member of the Tec family kinases, Bruton’s tyrosine kinase (Btk) modulates B-cell development and activation, and plays an important role in antibody production. Interestingly, Btk and Tec (the other Tec kinase family) regulate osteoclast (OC) differentiation via Receptor Activator of Nuclear Factor κ B (RANK) signaling. Moreover, OCs derived from X-linked agammaglobulinemia (XLA) patients who harbor Btk null mutations have impaired function. Here we show that a potent and specific Btk inhibitor, CC-292 inhibits OC function in multiple myeloma (MM) patients. CC-292 is a highly selective, covalent Btk inhibitor. OC derived from MM patient monocytes were assayed with or without CC-292. Interestingly, OC function was significantly inhibited in the presence of CC-292 (100 nM and 1000 nM) as demonstrated by pit formation assay. However, mRNA expression for TRAP and Cathepsin K, two OC differentiation markers were increased in the presence of CC-292 suggesting that CC-292 inhibits OC function without inhibiting OC differentiation. OC sealing zone contributes to OC bone resorption function. Given the role of c-Src and Proline-rich tyrosine Kinase 2 (Pyk2) signaling in sealing zone formation and OC function we next evaluated CC-292’s effect on Pyk2 and c-Src. Pyk2 plays a role in OC activation and localizes to the sealing zone in OC. RANK signaling activates c-Src, which phosphorylates Pyk2. Moreover c-Src controls OC bone resorption by regulating actin organization via cortactin. Interestingly, CC-292 (100 nM) inhibited c-Src total protein, c-Src phosphorylation and Pyk2 phosphorylation. Furthermore, CC-292 (100 nM) inhibited cortactin protein and mRNA expression, and upregulated c-Cbl protein (E3 ubiquitin ligase for c-Src) expression in OC derived from MM patient monocytes with resultant inhibition of OC sealing zone formation. However, at the same low doses (100 nM) CC-292 did not show any direct in vitro effect against MM cell viability. Because carfilzomib, a proteasome inhibitor that binds irreversibly to its target, has potent anti-MM activity and also inhibits OC resorptive activity, we studied CC-292 in combination with carfilzomib. Our data suggests that carfilzomib (1.25 nM) has no impact on OC sealing zone formation but inhibits OC differentiation. CC-292 (100 nM) in combination with carfilzomib (1.25 nM) inhibited not only sealing zone formation but also OC differentiation, resulting in stronger suppression of OC function than carfilzomib alone. The combination of CC-292 (30mg/kg p.o. for 5 days per week for 6 weeks) and carfilzomib (3 mg/kg i.v. x 2 days per week for 4 weeks and 2 mg/kg i.v. x 2 days per week for 2 weeks) significantly inhibited tumor burden and myeloma cell numbers in a diffuse NOD-SCID MM model. The calvarial cells derived from these mice treated with CC-292 alone, carfilzomib alone or the combination showed higher osteocalcin mRNA (osteoblast differentiation marker) expression. A specific bone resorption marker, carboxy-terminal telopeptide collagen crosslinks (CTX) in mouse serum was significantly inhibited in CC-292 and CC-292 in combination with carfilzomib treatment groups in comparison with control mice. Furthermore, 3D microCT reconstructions showed increase in cancellous bone volume in lumbar vertebrae in mice treated with CC-292 or carfilzomib, while the combination treatment resulted in an increase in cancellous bone volume in an additive manner. These data demonstrate that the novel BTK inhibitor CC-292 inhibits OC function through inhibition of OC sealing zone formation. Moreover, CC-292 in combination with carfilzomib augments effects against the bone microenvironment with resultant anti-MM activity. Disclosures: Arastu-Kapur: Onyx Pharmaceuticals, Inc.: Employment. Evans:Celgene Avilomics Research: Employment, Equity Ownership. Singh:Celgene Avilomics Research: Employment, Equity Ownership. Kirk:Onyx Pharmaceuticals, Inc.: Employment. Westlin:Celgene Avilomics Research: Employment, Equity Ownership. Raje:Celgene: Consultancy; Millenium: Consultancy; Onyx: Consultancy; Amgen: Consultancy; Acetylon: Research Funding; Eli Lilly: Research Funding.

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Apatite-mediated Actin Dynamics in Resorbing Osteoclasts

Molecular Biology of the Cell ◽

10.1091/mbc.e04-06-0522 ◽

2004 ◽

Vol 15 (12) ◽

pp. 5231-5241 ◽

Cited By ~ 191

Author(s):

Frédéric Saltel ◽

Olivier Destaing ◽

Frédéric Bard ◽

Diane Eichert ◽

Pierre Jurdic

Keyword(s):

Bone Resorption ◽

Actin Dynamics ◽

Cell Periphery ◽

Main Function ◽

Zone Formation ◽

Actin Structure ◽

Sealing Zone ◽

Mineralized Matrix ◽

And Migration

The actin cytoskeleton is essential for osteoclasts main function, bone resorption. Two different organizations of actin have been described in osteoclasts, the podosomes belt corresponding to numerous F-actin columns arranged at the cell periphery, and the sealing zone defined as a unique large band of actin. To compare the role of these two different actin organizations, we imaged osteoclasts on various substrata: glass, dentin, and apatite. Using primary osteoclasts expressing GFP-actin, we found that podosome belts and sealing zones, both very dynamic actin structures, were present in mature osteoclasts; podosome belts were observed only in spread osteoclasts adhering onto glass, whereas sealing zone were seen in apico-basal polarized osteoclasts adherent on mineralized matrix. Dynamic observations of several resorption cycles of osteoclasts seeded on apatite revealed that 1) podosomes do not fuse together to form the sealing zone; 2) osteoclasts alternate successive stationary polarized resorption phases with a sealing zone and migration, nonresorption phases without any specific actin structure; and 3) apatite itself promotes sealing zone formation though c-src and Rho signaling. Finally, our work suggests that apatite-mediated sealing zone formation is dependent on both c-src and Rho whereas apico-basal polarization requires only Rho.

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AVL-292, a Bruton's Tyrosine Kinase Inhibitor Impacts Bone Resorption by Abrogating Osteoclast Sealing Zone Formation in Multiple Myeloma

Blood ◽

10.1182/blood.v120.21.1822.1822 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1822-1822

Author(s):

Homare Eda ◽

Loredana Santo ◽

Diana Cirstea ◽

Andrew J. Yee ◽

Anuj Mahindra ◽

...

Keyword(s):

Bone Resorption ◽

Tyrosine Kinase ◽

Mrna Expression ◽

Mechanism Of Action ◽

Research Funding ◽

Bruton's Tyrosine Kinase ◽

Zone Formation ◽

Sealing Zone ◽

Btk Inhibitor ◽

Rank Signaling

Abstract Abstract 1822 The activation of Bruton's tyrosine kinase (Btk), a member of the Tec family of tyrosine kinases regulates B-cell development and activation, and plays a key role in antibody production. Interestingly, Btk and the other Tec kinase family, Tec, regulate OC differentiation via Receptor Activator of Nuclear Factor κ B (RANK) signaling. Moreover, patients with X-linked agammaglobulinemia (XLA) who harbor Btk null mutations have impaired OC function. Here we show that, a potent and specific inhibitor of Btk, AVL-292, inhibits OC function in MM patients. AVL-292 is a highly selective, covalent Btk inhibitor that potently silences Btk enzymatic activity (IC50 < 0.5nM) with high selectivity towards Btk with lack of significant inhibition against other kinases involved in BCR signaling (Syk, Lyn). We examined the mechanism of action of AVL-292 in the context of OCs function. OC derived from MM patient monocytes were assayed with or without AVL-292. Interestingly, OC function was inhibited in the presence AVL-292 as demonstrated by pit formation assay. However, mRNA expression of Cathepsin K and TRAP, markers of OC differentiation, were increased in the presence of AVL-292. These data suggest AVL-292 inhibits OC function without affecting the OC differentiation. It has been shown that BTK and Tec regulation of OC differentiation is related to calcium (Ca2+) signaling by increasing Ca2+ flux through direct phosphorylation of phospholipase C2 (PLCγ2). To delineate the mechanism of action of the BTK inhibitor against OC function, the RANK signaling proteins were detected by western blotting and Ca2+ concentration was measured by flourescence. AVL-292 inhibited phosphorylation of the BTK substrate, PLCγ2 in OCs. This was associated with an inhibition of intracellular Ca2+ release by AVL-292 which otherwise increased with RANKL stimulation in OCs. Given the effect of AVL-292 on RANK signaling we next evaluated its effect on Proline-rich tyrosine Kinase 2 (Pyk2) and c-Src. Pyk2 plays a role in OC activation and localizes to the sealing zone in OC. RANK signaling activates c-Src, which phosphorylates Pyk2. Moreover c-Src controls OC bone resorption by regulating actin organization via cortactin. Interestingly, AVL-292 inhibited c-Src and Pyk2 phosphorylation. Furthermore, AVL-292 inhibited cortactin protein and mRNA expression, and upregulated c-Cbl protein (E3 ubiquitin ligase for c-Src) expression in OCs derived from MM patient monocytes. These data demonstrate that the novel BTK inhibitor AVL-292 inhibits OC function through inhibition of OC sealing zone formation. These results suggest a potential novel mechanism of Btk inhibition with AVL-292 on osteoclast function and therefore bone resorption. Disclosures: Evans: Celgene: Employment. Singh:Celgene: Employment. Westlin:Celgene: Employment. Raje:Onyx: Consultancy; Celgene: Consultancy; Millenium: Consultancy; Acetylon: Research Funding; Amgen: Research Funding; Eli-Lilly: Research Funding.

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Regulation of bone resorption and sealing zone formation in osteoclasts occurs through protein kinase b-mediated microtubule stabilization

Journal of Bone and Mineral Research ◽

10.1002/jbmr.1844 ◽

2013 ◽

Vol 28 (5) ◽

pp. 1191-1202 ◽

Cited By ~ 21

Author(s):

Takumi Matsumoto ◽

Yuichi Nagase ◽

Jun Hirose ◽

Naoto Tokuyama ◽

Tetsuro Yasui ◽

...

Keyword(s):

Protein Kinase ◽

Bone Resorption ◽

Protein Kinase B ◽

Zone Formation ◽

Microtubule Stabilization ◽

Sealing Zone

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Defective microtubule-dependent podosome organization in osteoclasts leads to increased bone density in Pyk2−/− mice

The Journal of Cell Biology ◽

10.1083/jcb.200701148 ◽

2007 ◽

Vol 178 (6) ◽

pp. 1053-1064 ◽

Cited By ~ 163

Author(s):

Hava Gil-Henn ◽

Olivier Destaing ◽

Natalie A. Sims ◽

Kazuhiro Aoki ◽

Neil Alles ◽

...

Keyword(s):

Bone Resorption ◽

Protein Tyrosine Kinase ◽

Ectopic Expression ◽

Cell Periphery ◽

Wild Type ◽

Zone Formation ◽

Sealing Zone ◽

Osteoclast Function ◽

Actin Rings

The protein tyrosine kinase Pyk2 is highly expressed in osteoclasts, where it is primarily localized in podosomes. Deletion of Pyk2 in mice leads to mild osteopetrosis due to impairment in osteoclast function. Pyk2-null osteoclasts were unable to transform podosome clusters into a podosome belt at the cell periphery; instead of a sealing zone only small actin rings were formed, resulting in impaired bone resorption. Furthermore, in Pyk2-null osteoclasts, Rho activity was enhanced while microtubule acetylation and stability were significantly reduced. Rescue experiments by ectopic expression of wild-type or a variety of Pyk2 mutants in osteoclasts from Pyk2−/− mice have shown that the FAT domain of Pyk2 is essential for podosome belt and sealing zone formation as well as for bone resorption. These experiments underscore an important role of Pyk2 in microtubule-dependent podosome organization, bone resorption, and other osteoclast functions.

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The cell biology of osteoclast function

Journal of Cell Science ◽

10.1242/jcs.113.3.377 ◽

2000 ◽

Vol 113 (3) ◽

pp. 377-381 ◽

Cited By ~ 24

Author(s):

H.K. Vaananen ◽

H. Zhao ◽

M. Mulari ◽

J.M. Halleen

Keyword(s):

Bone Resorption ◽

Cell Biology ◽

Degradation Products ◽

Bone Matrix ◽

Organic Matrix ◽

Endosomal Membrane ◽

Multinucleated Cells ◽

Sealing Zone ◽

Ruffled Border ◽

Osteoclast Function

Osteoclasts are multinucleated cells responsible for bone resorption. They have developed an efficient machinery for dissolving crystalline hydroxyapatite and degrading organic bone matrix rich in collagen fibers. When initiating bone resorption, osteoclasts become polarized, and three distinct membrane domains appear: a ruffled border, a sealing zone and a functional secretory domain. Simultaneously, the cytoskeleton undergoes extensive re-organisation. During this process, the actin cytoskeleton forms an attachment ring at the sealing zone, the membrane domain that anchors the resorbing cell to bone matrix. The ruffled border appears inside the sealing zone, and has several characteristics of late endosomal membrane. Extensive vesicle transport to the ruffled border delivers hydrochloric acid and proteases to an area between the ruffled border and the bone surface called the resorption lacuna. In this extracellular compartment, crystalline hydroxyapatite is dissolved by acid, and a mixture of proteases degrades the organic matrix. The degradation products of collagen and other matrix components are endocytosed, transported through the cell and exocytosed through a functional secretory domain. This transcytotic route allows osteoclasts to remove large amounts of matrix-degradation products without losing their tight attachment to underlying bone. It also facilitates further processing of the degradation products intracellularly during the passage through the cell.

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Vitronectin receptor has a role in bone resorption but does not mediate tight sealing zone attachment of osteoclasts to the bone surface.

The Journal of Cell Biology ◽

10.1083/jcb.115.4.1179 ◽

1991 ◽

Vol 115 (4) ◽

pp. 1179-1186 ◽

Cited By ~ 113

Author(s):

P T Lakkakorpi ◽

M A Horton ◽

M H Helfrich ◽

E K Karhukorpi ◽

H K Väänänen

Keyword(s):

Bone Resorption ◽

Cell Membrane ◽

Immunoperoxidase Staining ◽

Bone Surface ◽

Intense Staining ◽

Vitronectin Receptor ◽

Gold Particles ◽

Light Microscopical Level ◽

Sealing Zone ◽

Arginine Glycine Aspartic Acid

During bone resorption, osteoclasts form a tight attachment, the sealing zone, around resorption lacunae. Vitronectin receptor has previously been shown to be expressed in osteoclasts and it has been suggested that it mediates the tight attachment at the sealing zone. In this study we have shown that glycine-arginine-glycine-aspartic acid-serine pentapeptide inhibits bone resorption by isolated osteoclasts and drastically changes the morphology of the osteoclasts. When the vitronectin receptor was localized by immunofluorescence in rat and chicken osteoclasts cultured on bone slices, it was found to be distributed throughout the osteoclast cell membrane except in the sealing zone areas. Immunoperoxidase staining of rat bone sections at the light microscopical level also revealed intense staining of the cell membrane with occasional small unstained areas, probably corresponding to the sealing zones. Immunoelectron microscopy confirmed the results obtained by light microscopy showing specific labeling only at the ruffled borders and basolateral membranes (0.82 and 2.43 gold particles/microns of membrane, respectively), but not at the sealing zone areas (0.06 gold particles/microns of membrane). Both alpha v and beta 3 subunits of the vitronectin receptor were similarly localized. These results strongly suggest that, although the vitronectin receptor is important in the function of osteoclasts, it is not mediating the final sealing zone attachment of the osteoclasts to the mineralized bone surface.

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COMPLETE SEALING ZONE FORMATION NEEDS MULTIPLE GENE PARTICIPATION

Bioceramics ◽

10.1142/9789814291064_0066 ◽

1999 ◽

Author(s):

Reiko Honma ◽

Wei Chen ◽

Yin-kun Liu ◽

Hua-yun Deng ◽

Atsuko Nemoto ◽

...

Keyword(s):

Zone Formation ◽

Multiple Gene ◽

Sealing Zone

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Dock5 is a new regulator of microtubule dynamic instability through GSK3β inhibition in osteoclasts

10.1101/653055 ◽

2019 ◽

Author(s):

Sarah Guimbal ◽

David Guérit ◽

Manon Chardon ◽

Anne Blangy ◽

Virginie Vives

Keyword(s):

Dynamic Instability ◽

Direct Interaction ◽

Background Information ◽

Growth Speed ◽

Microtubule Dynamic ◽

Zone Formation ◽

Acetylated Tubulin ◽

Bone Degradation ◽

Sealing Zone ◽

Osteoclast Resorption

AbstractBackground informationOsteoclast resorption is dependent on a podosome-rich structure, called sealing zone, which is stabilized by acetylated microtubules. It tightly attaches the osteoclast to the bone creating a favorable acidic microenvironment for bone degradation. We already established that Rac activation by Dock5 is necessary for osteoclast resorption. Indeed, inhibition of Dock5 in osteoclasts results in Rac1 decreased activity associated to impaired podosome assembly into sealing zones and resorbing activity.ResultsIn this report, we show that Dock5 knockout osteoclasts also present a reduced acetylated tubulin level leading to a decreased length and duration of microtubule growth phases whereas their growth speed remains unaffected. Dock5 does not act by direct interaction with the polymerized tubulin but through inhibition of the microtubules destabilizing kinase GSK3β downstream of Akt activation. Interestingly, we ruled out the implication of Rac1 in this process using specific inhibitors.ConclusionOur data involve Dock5 as a new regulator of microtubule dynamic instability in osteoclast.SignificanceThe fact that Dock5 is a regulator of both actin cytoskeleton and microtubule dynamics makes it an interesting therapeutic target for osteolytic pathologies because of its dual role on sealing zone formation and stabilization.

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