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

2007 ◽  
Vol 178 (6) ◽  
pp. 1053-1064 ◽  
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.

scholarly journals Apatite-mediated Actin Dynamics in Resorbing Osteoclasts

2004 ◽  
Vol 15 (12) ◽  
pp. 5231-5241 ◽  
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.

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

1999 ◽  
Vol 112 (22) ◽  
pp. 3985-3993 ◽  
Author(s):  
I. Nakamura ◽  
M.F. Pilkington ◽  
P.T. Lakkakorpi ◽  
L. Lipfert ◽  
S.M. Sims ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Cell Attachment ◽  
Leading Edge ◽  
In Vivo Studies ◽  
Sealing Zone ◽  
Osteoclast Function ◽  
Alpha V Beta 3

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).

scholarly journals Osteopontin Deficiency Produces Osteoclast Dysfunction Due to Reduced CD44 Surface Expression

2003 ◽  
Vol 14 (1) ◽  
pp. 173-189 ◽  
Author(s):  
M. A. Chellaiah ◽  
N. Kizer ◽  
R. Biswas ◽  
U. Alvarez ◽  
J. Strauss-Schoenberger ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Surface Expression ◽  
Wild Type ◽  
Exogenous Addition ◽  
Load To Failure ◽  
Ruffled Border ◽  
Osteoclast Function ◽  
Deficient Mice

Osteopontin (OPN) was expressed in murine wild-type osteoclasts, localized to the basolateral, clear zone, and ruffled border membranes, and deposited in the resorption pits during bone resorption. The lack of OPN secretion into the resorption bay of avian osteoclasts may be a component of their functional resorption deficiency in vitro. Osteoclasts deficient in OPN were hypomotile and exhibited decreased capacity for bone resorption in vitro. OPN stimulated CD44 expression on the osteoclast surface, and CD44 was shown to be required for osteoclast motility and bone resorption. Exogenous addition of OPN to OPN−/− osteoclasts increased the surface expression of CD44, and it rescued osteoclast motility due to activation of the αvβ3 integrin. Exogenous OPN only partially restored bone resorption because addition of OPN failed to produce OPN secretion into resorption bays as seen in wild-type osteoclasts. As expected with these in vitro findings of osteoclast dysfunction, a bone phenotype, heretofore unappreciated, was characterized in OPN-deficient mice. Delayed bone resorption in metaphyseal trabeculae and diminished eroded perimeters despite an increase in osteoclast number were observed in histomorphometric measurements of tibiae isolated from OPN-deficient mice. The histomorphometric findings correlated with an increase in bone rigidity and moment of inertia revealed by load-to-failure testing of femurs. These findings demonstrate the role of OPN in osteoclast function and the requirement for OPN as an osteoclast autocrine factor during bone remodeling.

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

2000 ◽  
Vol 268 (1) ◽  
pp. 201-209 ◽  
Author(s):  
Cécile Itzstein ◽  
Léon Espinosa ◽  
Pierre D. Delmas ◽  
Chantal Chenu
Keyword(s):  
Bone Resorption ◽  
Nmda Receptors ◽  
Zone Formation ◽  
Sealing Zone

The cell biology of osteoclast function

2000 ◽  
Vol 113 (3) ◽  
pp. 377-381 ◽  
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.

scholarly journals Free Fatty Acid Receptor 4 (GPR120) Stimulates Bone Formation and Suppresses Bone Resorption in the Presence of Elevated n-3 Fatty Acid Levels

Endocrinology ◽  
2016 ◽  
Vol 157 (7) ◽  
pp. 2621-2635 ◽  
Author(s):  
Seong Hee Ahn ◽  
Sook-Young Park ◽  
Ji-Eun Baek ◽  
Su-Youn Lee ◽  
Wook-Young Baek ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Free Fatty Acid ◽  
Bone Resorption ◽  
Bone Loss ◽  
Bone Formation ◽  
High Fat Diet ◽  
Wild Type ◽  
High Fat ◽  
Free Fatty Acid Receptor

Free fatty acid receptor 4 (FFA4) has been reported to be a receptor for n-3 fatty acids (FAs). Although n-3 FAs are beneficial for bone health, a role of FFA4 in bone metabolism has been rarely investigated. We noted that FFA4 was more abundantly expressed in both mature osteoclasts and osteoblasts than their respective precursors and that it was activated by docosahexaenoic acid. FFA4 knockout (Ffar4−/−) and wild-type mice exhibited similar bone masses when fed a normal diet. Because fat-1 transgenic (fat-1Tg+) mice endogenously converting n-6 to n-3 FAs contain high n-3 FA levels, we crossed Ffar4−/− and fat-1Tg+ mice over two generations to generate four genotypes of mice littermates: Ffar4+/+;fat-1Tg−, Ffar4+/+;fat-1Tg+, Ffar4−/−;fat-1Tg−, and Ffar4−/−;fat-1Tg+. Female and male littermates were included in ovariectomy- and high-fat diet-induced bone loss models, respectively. Female fat-1Tg+ mice decreased bone loss after ovariectomy both by promoting osteoblastic bone formation and inhibiting osteoclastic bone resorption than their wild-type littermates, only when they had the Ffar4+/+ background, but not the Ffar4−/− background. In a high-fat diet-fed model, male fat-1Tg+ mice had higher bone mass resulting from stimulated bone formation and reduced bone resorption than their wild-type littermates, only when they had the Ffar4+/+ background, but not the Ffar4−/− background. In vitro studies supported the role of FFA4 as n-3 FA receptor in bone metabolism. In conclusion, FFA4 is a dual-acting factor that increases osteoblastic bone formation and decreases osteoclastic bone resorption, suggesting that it may be an ideal target for modulating metabolic bone diseases.

A Novel Bruton′s Tyrosine Kinase Inhibitor CC-292 In Combination With The Proteasome Inhibitor Carfilzomib Iimpacts Multiple Myeloma Bone Microenviroment With Resultant Anti-Myeloma Activity

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 682-682 ◽  
Author(s):  
Homare Eda ◽  
Loredana Santo ◽  
Diana D Cirstea ◽  
Andrew J Yee ◽  
Tyler A Scullen ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Tyrosine Kinase ◽  
Proteasome Inhibitor ◽  
Bone Volume ◽  
Employment Equity ◽  
Zone Formation ◽  
Sealing Zone ◽  
Btk Inhibitor ◽  
Equity Ownership ◽  
Rank Signaling

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.

scholarly journals An Essential Role of Cytosolic Phospholipase A2α in Prostaglandin E2–mediated Bone Resorption Associated with Inflammation

2003 ◽  
Vol 197 (10) ◽  
pp. 1303-1310 ◽  
Author(s):  
Chisato Miyaura ◽  
Masaki Inada ◽  
Chiho Matsumoto ◽  
Tomoyasu Ohshiba ◽  
Naonori Uozumi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Resorption ◽  
Bone Loss ◽  
Stromal Cells ◽  
Osteoclast Formation ◽  
Wild Type ◽  
Cytosolic Phospholipase ◽  
Cytosolic Phospholipase A2α ◽  
Cox 2

Prostaglandin E (PGE)2 produced by osteoblasts acts as a potent stimulator of bone resorption. Inflammatory bone loss is accompanied by osteoclast formation induced by bone-resorbing cytokines, but the mechanism of PGE2 production and bone resorption in vivo is not fully understood. Using cytosolic phospholipase A2α (cPLA2α)-null mice, we examined the role of cPLA2α in PGE2 synthesis and bone resorption. In bone marrow cultures, interleukin (IL)-1 markedly stimulated PGE2 production and osteoclast formation in wild-type mice, but not in cPLA2α-null mice. Osteoblastic bone marrow stromal cells induced the expression of cyclooxygenase (COX)-2 and membrane-bound PGE2 synthase (mPGES) in response to IL-1 and lipopolysaccharide (LPS) to produce PGE2. Osteoblastic stromal cells collected from cPLA2α-null mice also induced the expression of COX-2 and mPGES by IL-1 and LPS, but could not produce PGE2 due to the lack of arachidonic acid release. LPS administration to wild-type mice reduced femoral bone mineral density by increased bone resorption. In cPLA2α-null mice, however, LPS-induced bone loss could not be observed at all. Here, we show that cPLA2α plays a key role in PGE production by osteoblasts and in osteoclastic bone resorption, and suggest a new approach to inflammatory bone disease by inhibiting cPLA2α.

scholarly journals Pseudomonas syringae Evades Host Immunity by Degrading Flagellin Monomers with Alkaline Protease AprA

2014 ◽  
Vol 27 (7) ◽  
pp. 603-610 ◽  
Author(s):  
Michiel J. C. Pel ◽  
Anja J. H. van Dijken ◽  
Bart W. Bardoel ◽  
Michael F. Seidl ◽  
Sjoerd van der Ent ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Alkaline Protease ◽  
Bacterial Species ◽  
Gene Activation ◽  
Ectopic Expression ◽  
Host Immunity ◽  
Wild Type ◽  
Knockout Mutant ◽  
Inhibitory Peptide

Bacterial flagellin molecules are strong inducers of innate immune responses in both mammals and plants. The opportunistic pathogen Pseudomonas aeruginosa secretes an alkaline protease called AprA that degrades flagellin monomers. Here, we show that AprA is widespread among a wide variety of bacterial species. In addition, we investigated the role of AprA in virulence of the bacterial plant pathogen P. syringae pv. tomato DC3000. The AprA-deficient DC3000 ΔaprA knockout mutant was significantly less virulent on both tomato and Arabidopsis thaliana. Moreover, infiltration of A. thaliana Col-0 leaves with DC3000 ΔaprA evoked a significantly higher level of expression of the defense-related genes FRK1 and PR-1 than did wild-type DC3000. In the flagellin receptor mutant fls2, pathogen virulence and defense-related gene activation did not differ between DC3000 and DC3000 ΔaprA. Together, these results suggest that AprA of DC3000 is important for evasion of recognition by the FLS2 receptor, allowing wild-type DC3000 to be more virulent on its host plant than AprA-deficient DC3000 ΔaprA. To provide further evidence for the role of DC3000 AprA in host immune evasion, we overexpressed the AprA inhibitory peptide AprI of DC3000 in A. thaliana to counteract the immune evasive capacity of DC3000 AprA. Ectopic expression of aprI in A. thaliana resulted in an enhanced level of resistance against wild-type DC3000, while the already elevated level of resistance against DC3000 ΔaprA remained unchanged. Together, these results indicate that evasion of host immunity by the alkaline protease AprA is important for full virulence of strain DC3000 and likely acts by preventing flagellin monomers from being recognized by its cognate immune receptor.

Modularity of osteoclast behaviour and “mode-specific” inhibition of osteoclast function

1990 ◽  
Vol 10 (6) ◽  
pp. 547-556 ◽  
Author(s):  
Mone Zaidi
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Bone Resorption ◽  
Specific Inhibition ◽  
Hydrogen Ions ◽  
Pharmacological Agents ◽  
Osteoclastic Activity ◽  
Osteoclast Function ◽  
Scanning Electron

This study is part of an attempt to understand the role of specific cellular activities in the bone resorptive process. Experiments were performed whereby known pharmacological agents were used to inhibit individual modes of osteoclastic activity, such as motility and secretion. The effects of such treatments on bone resorption were assessed by quantitative scanning electron microscopy. The compounds included colchicine, which was used to inhibit osteoclast motility; molybdate ions which were used to selectively inhibit the catalytic activity of secreted acid phosphatase, and omeprazole which was employed to inhibit the secretion of hydrogen ions. All compounds inhibited osteoclastic bone resorption, but singularly affected defined modes of activity. These findings suggest that each mode of osteoclastic activity is essential for the bone resorptive process, and that “mode-specific” inhibition may provide a means whereby excessive activity of the osteoclast can be regulated in disease.

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