Protein tyrosine phosphatases in osteoclast differentiation, adhesion, and bone resorption

Osteoclasts are large cells derived from the monocyte-macrophage hematopoietic cell lineage. Their primary function is to degrade bone in various physiological contexts. Osteoclasts adhere to bone via podosomes, specialized adhesion structures whose structure and subcellular organization are affected by mechanical contact of the cell with bone matrix. Ample evidence indicates that reversible tyrosine phosphorylation of podosomal proteins plays a major role in determining the organization and dynamics of podosomes. Although roles of several tyrosine kinases are known in detail in this respect, little is known concerning the roles of protein tyrosine phosphatases (PTPs) in regulating osteoclast adhesion. Here we summarize available information concerning the known and hypothesized roles of the best-researched PTPs in osteoclasts - PTPRO, PTP epsilon, SHP-1, and PTP-PEST. Of these, PTPRO, PTP epsilon, and PTP-PEST appear to support osteoclast activity while SHP-1 inhibits it. Additional studies are required to provide full molecular details of the roles of these PTPs in regulating osteoclast adhesion, and to uncover additional PTPs that participate in this process.     

Rac-GTPase, osteoclast cytoskeleton and bone resorption.

The members of the Rho-GTPase subfamily, Rac1 and Rac2, are intimately involved in the organization of the cytoskeleton, and the p21-activated kinases or PAKs are targets of these proteins. Rac1 and Rac2 are also essential components of NADPH oxidase, the enzyme responsible for generating free radicals. The cytoskeleton modulates the adhesion of osteoclasts to bone and its subsequent resorption. These cells contain NADPH diaphorase activity, and free radicals influence bone resorption. The influence of Rac1, Rac2 and PAK1 on the cytoskeleton, resorbing activity and NADPH diaphorase activity of disaggregated rat osteoclasts was investigated by permeabilisation with saponin and introducing specific anti-Rac1, anti-Rac2 or anti-PAK1 antibodies. Rhodamine-phalloidin stain was used to identify actin in osteoclasts cultured on plastic slides, and the bone-slice method was used to measure resorption. Saponin permeabilisation did not affect the cytoskeletal organization or bone resorption. Anti-Rac antibodies caused dose- and time-dependent cytoskeletal changes. The osteoclasts rounded up and developed retraction fibers; actin rings were disrupted and large actin dots were seen at the periphery of the cells. Osteoclast resorptive activity was depressed after incubation with the antibodies. The total area resorbed by treated cells and the mean pit area were smaller than those of controls. Anti-PAK1 antibody caused similar changes. None of the antibodies altered the NADPH diaphorase activity. Thus, Rac-GTPases are present in rat osteoclasts and are involved in the organization of the actin cytoskeleton and in resorptive activity. These effects may be mediated by PAK1 kinase, but do not influence osteoclast NADPH diaphorase activity.     

Human osteoclast ontogeny and pathological bone resorption

Monocytes and macrophages are capable of degrading both the mineral and organic components of bone and are known to secrete local factors which stimulate host osteoclastic bone resorption. Recent studies have shown that monocytes and macrophages, including those isolated from neoplastic and inflammatory lesions, can also be induced to differentiate into cells that show all the cytochemical and functional characteristics of mature osteoclasts, including lacunar bone resorption. Monocyte/macrophage-osteoclast differentiation occurs in the presence of osteoblasts/bone stromal cells (which express osteoclast differentiation factor) and macrophage-colony stimulating factor and is inhibited by osteoprotegerin. Various systemic hormones and local factors (e.g. cytokines, growth factors, prostaglandins) modulate osteoclast formation by controlling these cellular and humoral elements. Various pathological lesions of bone and joint (e.g. carcinomatous metastases, arthritis, aseptic loosening) are associated with osteolysis. These lesions generally contain a chronic inflammatory infiltrate in which macrophages form a significant fraction. One cellular mechanism whereby pathological bone resorption may be effected is through generation of increased numbers of bone-resorbing osteoclasts from macrophages. Production of humoral factors which stimulate mononuclear phagocyte-osteoclast differentiation and osteoclast activity is also likely to influence the extent of pathological bone resorption.     

Recent advances in osteoclast biology and pathological bone resorption

The osteoclast is a bone-degrading polykaryon. Recent studies have clarified the differentiation of this cell and the biochemical mechanisms it uses to resorb bone. The osteoclast derives from a monocyte/macrophage precursor. Osteoclast formation requires permissive concentrations of M-CSF and is driven by contact with mesenchymal cells in bone that bear the TNF-family ligand RANKL. Osteoclast precursors express RANK, and the interaction between RANKL and RANK (which is inhibited by OPG) is the major determinant of osteoclast formation. Hormones, such as PTH/PTHrP, glucocorticoids and 1,25(OH)2D3, and humoral factors, including TNFalpha, interleukin-1, TGFss and prostaglandins, influence osteoclast formation by altering expression of these molecular factors. TNFalpha, IL-6 and IL-11 have also been shown to promote osteoclast formation by RANKL-independent processes. RANKL-dependent/independent osteoclast formation is likely to play an important role in conditions where there is pathological bone resorption such as inflammatory arthritis and malignant bone resorption. Osteoclast functional defects cause sclerotic bone disorders, many of which have recently been identified as specific genetic defects. Osteoclasts express specialized proteins including a vacuolar-type H+-ATPase that drives HCl secretion for dissolution of bone mineral. One v-ATPase component, the 116 kD V0 subunit, has several isoforms. Only one isoform, TCIRG1, is up-regulated in osteoclasts. Defects in TCIRG1 are common causes of osteopetrosis. HCl secretion is dependent on chloride channels; a chloride channel homologue, CLCN7, is another common defect in osteopetrosis. Humans who are deficient in carbonic anhydrase II or who have defects in phagocytosis also have variable defects in bone remodelling. Organic bone matrix is degraded by thiol proteinases, principally cathepsin K, and abnormalities in cathepsin K cause another sclerotic bone disorder, pycnodysostosis. Thus, bone turnover in normal subjects depends on relative expression of key cytokines, and defects in osteoclastic turnover usually reflect defects in specific ion transporters or enzymes that play essential roles in bone degradation.     

Rho-A is critical for osteoclast podosome organization, motility, and bone resorption

Rho plays a regulatory role in the formation of actin stress fibers and focal adhesions, and it is also involved in integrin-mediated signaling events. To study the role of Rho in alpha(v)beta(3)/gelsolin-dependent signaling, the HIV-Tat peptide, hemagglutinin (HA)-tagged Rho(Val-14) (constitutively active) and Rho(Asn-19) (dominant negative) were transduced into avian osteoclasts. Protein transduction by HA-Tat was highly efficient, and 90-100% of the cells were transduced with HA-tagged proteins. We demonstrate here that Rho(Val-14) transduction (100 nM) stimulated gelsolin-associated phosphatidylinositol 3-kinase activity, podosome assembly, stress fiber formation, osteoclast motility, and bone resorption, mimicking osteoclast stimulation by osteopontin/alpha(v)beta(3.) The effects of Rho(Val-14) transduction stimulation was time-dependent. C3 exoenzyme blocked the effects of Rho(Val-14) and induced podosome disassembly, loss of motility, and inhibition of bone resorption. Transduction of Rho(Asn-19) produced podosome disassembly, and blocked osteopontin stimulation. These data demonstrate that integrin-dependent activation of phosphoinositide synthesis, actin stress fiber formation, podosome reorganization for osteoclast motility, and bone resorption require Rho stimulation.     

Polyphosphoinositides-dependent regulation of the osteoclast actin cytoskeleton and bone resorption

Background  

Gelsolin, an actin capping protein of osteoclast podosomes, has a unique function in regulating assembly and disassembly of the podosome actin filament. Previously, we have reported that osteopontin (OPN) binding to integrin α_vβ₃ increased the levels of gelsolin-associated polyphosphoinositides, podosome assembly/disassembly, and actin filament formation. The present study was undertaken to identify the possible role of polyphosphoinositides and phosphoinositides binding domains (PBDs) of gelsolin in the osteoclast cytoskeletal structural organization and osteoclast function.     

Cell adhesion is a prerequisite for osteoclast survival

The present study demonstrates that loss of cell adhesion potently promotes apoptosis in osteoclasts, a process termed "anoikis." When purified mature rabbit osteoclasts were cultured on plastic for 18 h, about 25% of them were spontaneously committed to apoptosis. The death rate increased more than twofold, after osteoclasts were subjected to suspension culture in inverted Terasaki plates. The osteoclast anoikis was significantly prevented by bongkrekic acid, an inhibitor of mitochondrial permeability transition (PT), and z-VAD-FMK, a caspase inhibitor, suggesting involvement of mitochondrial PT and caspase activation in the death process. Colony-stimulating factor-1 (CSF-1), receptor activator of NF-kappaB ligand (RANKL), and calcitonin protected adherent osteoclasts, but not floating osteoclasts from anoikis. These data show that adhesion is a primary requirement for osteoclast survival.     

Downregulation of small GTPase Rab7 impairs osteoclast polarization and bone resorption

During skeletal growth and remodeling the mineralized bone matrix is resorbed by osteoclasts through the constant secretion of protons and proteases to the bone surface. This relies on the formation of specialized plasma membrane domains, the sealing zone and the ruffled border, and vectorial transportation of intracellular vesicles in bone-resorbing osteoclasts. Here we show that Rab7, a small GTPase that is associated with late endosomes, is highly expressed and is predominantly localized at the ruffled border in bone-resorbing osteoclasts. The decreased expression of Rab7 in cultured osteoclasts by antisense oligodeoxynucleotides disrupted the polarization of the osteoclasts and the targeting of vesicles to the ruffled border. These impairments caused a significant inhibition of bone resorption in vitro. The results indicate that the late endocytotic pathway is involved in the osteoclast polarization and bone resorption and underscore the importance of Rab7 in osteoclast function.     

Co-culture with endothelial progenitor cells promotes survival,migration, and differentiation of osteoclast precursors

In this study, we report the effect of endothelial progenitor cells (EPCs) on the biological behavior of osteoclast precursors in vitro by establishing an indirect co-culture system of mice EPCs and RAW 264.7 monocyte cells. Results show that the survival, migration, and differentiation of osteoclast precursors were greatly enhanced when co-cultured with EPCs. These phenotypic changes coincide with the upregulation of multiple genes affected cell behavior, including phospho-VEGFR-2, CXCR4, phospho-Smad2/3, phospho-Akt, phospho-ERK1, and phospho-p38 MAPK. The results collectively suggest that EPCs could modulate the survival, migration, and differentiation potential of osteoclast precursors, thus providing new insights in understanding of correlation between angiogenesis and bone homeostasis.     

Stimulation of bone resorption and osteoclast clear zone formation by low pH: A time-course study

The significance of low pH-induced stimulation of osteoclastic bone resorption has recently been questioned following the finding that embryonic chick osteoclasts were only weakly stimulated by extremely low pH (6.5) and that the effect was transient, apparently due to cytotoxicity. Although low pH in the range 6.8–7.2 is known to stimulate rat osteoclasts over 24 h, the long-term effects of low pH on mammalian osteoclasts are not known. We have therefore conducted time-course studies over 72 h on the effect of pH in the range 6.3–7.3 on bone resorption and cytotoxicity in both rat and chick osteoclasts. In neonatal rat osteoclasts, lowering extracellular pH produced a powerful and significant stimulation of resorption over 24 h. Detailed analysis of the resorption focus revealed that this was due mainly to a higher proportion of active osteoclasts at lower pH. In addition, osteoclasts excavated slightly larger pits at low pH. Stimulation was no longer significant at 72 h, however, due to a pH-dependent slowing of resorption at acid pH associated 1) with cytotoxicity primarily of nonosteoclastic cells and 2) with an acceleration of bone resorption after 24 h at more alkaline pH. Resorption stimulated by low pH was associated with the formation of actin-rich “clear zones” within the osteoclast. Chick osteoclasts were less sensitive to low pH than rat osteoclasts but nonetheless showed a consistently higher level of resorption at low pH over 24–72 h. These results suggest that protons play an important regulatory role in neonatal rat osteoclasts, and stimulate the formation of clear zones. The lower sensitivity of the chick osteoclast to acid pH may be due to a species difference or the chick osteoclast's higher basal level of resorption. © 1993 Wiley-Liss, Inc.     

c-Src control of chloride channel support for osteoclast HCl transport and bone resorption

Bone degradation by osteoclasts depends upon active transport of hydrogen ions to solubilize bone mineral. This transport is supported by the parallel actions of a proton ATPase and a chloride channel located in the osteoclast ruffled membrane. We have previously identified a novel chloride channel, p62, which appears to be the avian counterpart to CLIC-5b and is expressed coincident with the appearance of acid secretion as avian osteoclasts differentiate in culture. In this article, we show that suppression of CLIC-5b in differentiating avian osteoclasts results in decreased acidification by vesicles derived from these cells and decreased ability of the cells to resorb bone. Acidification is rescued by the presence of valinomycin, consistent with a selective loss of chloride channel but not proton pump activity. Osteoclast bone resorption is known to be dependent on the expression of the tyrosine kinase, c-Src. We show that CLIC-5b from osteoclasts has affinity for both Src SH2 and SH3 domains. We find that suppression of expression of Src in developing osteoclasts results in decreased vesicular acidification, which is rescued by valinomycin, consistent with the loss of chloride conductance in the proton pump-containing vesicles. Suppression of c-Src causes no change in the steady state level of CLIC-5b expression, but does result in failure of proton pump and CLIC-5b to colocalize in cultured osteoclast precursors. We conclude that suppression of c-Src interferes with osteoclast bone resorption by disrupting functional co-localization of proton pump and CLIC-5b.     

Retinoids stimulate periosteal bone resorption by enhancing the protein RANKL, a response inhibited by monomeric glucocorticoid receptor

Increased vitamin A (retinol) intake has been suggested to increase bone fragility. In the present study, we investigated effects of retinoids on bone resorption in cultured neonatal mouse calvarial bones and their interaction with glucocorticoids (GC). All-trans-retinoic acid (ATRA), retinol, retinalaldehyde, and 9-cis-retinoic acid stimulated release of (45)Ca from calvarial bones. The resorptive effect of ATRA was characterized by mRNA expression of genes associated with osteoclast differentiation, enhanced osteoclast number, and bone matrix degradation. In addition, the RANKL/OPG ratio was increased by ATRA, release of (45)Ca stimulated by ATRA was blocked by exogenous OPG, and mRNA expression of genes associated with bone formation was decreased by ATRA. All retinoid acid receptors (RARα/β/γ) were expressed in calvarial bones. Agonists with affinity to all receptor subtypes or specifically to RARα enhanced the release of (45)Ca and mRNA expression of Rankl, whereas agonists with affinity to RARβ/γ or RARγ had no effects. Stimulation of Rankl mRNA by ATRA was competitively inhibited by the RARα antagonist GR110. Exposure of calvarial bones to GC inhibited the stimulatory effects of ATRA on (45)Ca release and Rankl mRNA and protein expression. This inhibitory effect was reversed by the glucocorticoid receptor (GR) antagonist RU 486. Increased Rankl mRNA stimulated by ATRA was also blocked by GC in calvarial bones from mice with a GR mutation that blocks dimerization (GR(dim) mice). The data suggest that ATRA enhances periosteal bone resorption by increasing the RANKL/OPG ratio via RARα receptors, a response that can be inhibited by monomeric GR.     

Evidence that failure of osteoid bone matrix resorption is caused by perturbation of osteoclast polarization

Osteoclasts resorb bone by a complex dynamic process that initially involves attachment, polarization and enzyme secretion, followed by their detachment and migration to new sites. In this study, we postulated that mineralized and osteoid bone matrix signal osteoclasts differently, resulting in the resorption of mineralized bone matrix only. We, therefore, compared the cytoplasmic distribution of cytoskeletal proteins F-actin and vinculin using confocal laser-scanning microscopy in osteoclasts cultured on mineralized and demineralized bone slices and correlated the observations with their functional activity. Our results have demonstrated significant differences in F-actin and vinculin staining patterns between osteoclasts cultured on mineralized bone matrix and those on demineralized bone matrix. In addition, the structural variations were accompanied by significant differences in bone resorbing activity between osteoclasts grown on mineralized bone matrix and those on demineralized bone matrix after 24 h of culture -- resorption only occurring in mineralized bone but not in demineralized bone. These results indicated that failure of osteoid bone resorption is caused by perturbation of osteoclast polarization. © 1998 Chapman & Hall     

Effect of ormeloxifene on ovariectomy-induced bone resorption, osteoclast differentiation and apoptosis and TGF beta-3 expression

Effect of ormeloxifene, a multifunctional selective estrogen receptor modulator, on prevention of ovariectomy-induced bone resorption in retired breeder female rats, osteoclastogenesis using bone marrow cells from adult Balb/c mice cultured in presence of M-CSF and RANKL, osteoclast apoptosis using terminal deoxynucleotidyl transferase fragment end labeling and TGF beta-3 expression were investigated. Raloxifene, a benzothiophene reported to mimic effects of estrogen in bone, and estradiol were used for comparison. Ormeloxifene (10⁻⁶ and 10⁻⁸ M) significantly inhibited osteoclastogenesis (P < 0.001 versus vehicle control) as evidenced by lower number of TRAP-positive osteoclasts in bone marrow cultures and caused apoptosis of osteoclasts. The effect was almost equivalent to that observed in presence of estradiol-17 beta, except that significant number of cells undergoing apoptosis was evident even at 10⁻⁹ M concentration of estradiol-17 beta (P < 0.001). Raloxifene, though inhibited osteoclastogenesis at much lower concentrations (10⁻⁸ to 10⁻¹² M; P < 0.001), failed to cause apoptosis of osteoclasts at any of the concentrations used. While ormeloxifene, raloxifene and ethynylestradiol significantly prevented ovariectomy-induced bone loss in vivo in retired breeder female rats, prevention of ovariectomy-induced decrease in BMD and trabecular network of proximal tibia, calcium and phosphorus levels in femur and tibia and prevention of ovariectomy-induced down-regulation of TGF beta-3 expression in lumbar vertebrae was of lower order in raloxifene- than ormeloxifene- or ethynylestradiol-supplemented females. Both the SERMs, however, produced considerable estrogenic effects at the uterine level as evidenced by increase in weight, total and endometrial area and luminal epithelial cell height; the effect being generally greater in raloxifene- than ormeloxifene-treated rats. Findings demonstrate that inhibition of estrogen-deficiency osteoporosis by ormeloxifene, as in case of estradiol, was mediated via inhibition of osteoclastogenesis, apoptosis of osteoclasts and up-regulation of TGF beta-3 expression. Raloxifene, though effective in inhibiting osteoclastogenesis in vitro at much lower concentrations, was not only less potent in preventing ovariectomy-induced bone loss in retired breeder female rats in vivo but also appeared to have a different mechanism of action than ormeloxifene and estradiol.     

Regulation of cell migration and survival by focal adhesion targeting of Lasp-1

Large-scale proteomic and functional analysis of isolated pseudopodia revealed the Lim, actin, and SH3 domain protein (Lasp-1) as a novel protein necessary for cell migration, but not adhesion to, the extracellular matrix (ECM). Lasp-1 is a ubiquitously expressed actin-binding protein with a unique domain configuration containing SH3 and LIM domains, and is overexpressed in 8-12% of human breast cancers. We find that stimulation of nonmotile and quiescent cells with growth factors or ECM proteins facilitates Lasp-1 relocalization from the cell periphery to the leading edge of the pseudopodium, where it associates with nascent focal complexes and areas of actin polymerization. Interestingly, although Lasp-1 dynamics in migratory cells occur independently of c-Abl kinase activity and tyrosine phosphorylation, c-Abl activation by apoptotic agents specifically promotes phosphorylation of Lasp-1 at tyrosine 171, which is associated with the loss of Lasp-1 localization to focal adhesions and induction of cell death. Thus, Lasp-1 is a dynamic focal adhesion protein necessary for cell migration and survival in response to growth factors and ECM proteins.     

Prevention of wear particle-induced osteolysis by a novel V-ATPase inhibitor saliphenylhalamide through inhibition of osteoclast bone resorption

Wear particle-induced peri-implant loosening (Aseptic prosthetic loosening) is one of the most common causes of total joint arthroplasty. It is well established that extensive bone destruction (osteolysis) by osteoclasts is responsible for wear particle-induced peri-implant loosening. Thus, inhibition of osteoclastic bone resorption should prevent wear particle induced osteolysis and may serve as a potential therapeutic avenue for prosthetic loosening. Here, we demonstrate for the first time that saliphenylhalamide, a new V-ATPase inhibitor attenuates wear particle-induced osteolysis in a mouse calvarial model. In vitro biochemical and morphological assays revealed that the inhibition of osteolysis is partially attributed to a disruption in osteoclast acidification and polarization, both a prerequisite for osteoclast bone resorption. Interestingly, the V-ATPase inhibitor also impaired osteoclast differentiation via the inhibition of RANKL-induced NF-κB and ERK signaling pathways. In conclusion, we showed that saliphenylhalamide affected multiple physiological processes including osteoclast differentiation, acidification and polarization, leading to inhibition of osteoclast bone resorption in vitro and wear particle-induced osteolysis in vivo. The results of the study provide proof that the new generation V-ATPase inhibitors, such as saliphenylhalamide, are potential anti-resorptive agents for treatment of peri-implant osteolysis.     

Regulation of bone mass, bone loss and osteoclast activity by cannabinoid receptors

Accelerated osteoclastic bone resorption has a central role in the pathogenesis of osteoporosis and other bone diseases. Identifying the molecular pathways that regulate osteoclast activity provides a key to understanding the causes of these diseases and to the development of new treatments. Here we show that mice with inactivation of cannabinoid type 1 (CB1) receptors have increased bone mass and are protected from ovariectomy-induced bone loss. Pharmacological antagonists of CB1 and CB2 receptors prevented ovariectomy-induced bone loss in vivo and caused osteoclast inhibition in vitro by promoting osteoclast apoptosis and inhibiting production of several osteoclast survival factors. These studies show that the CB1 receptor has a role in the regulation of bone mass and ovariectomy-induced bone loss and that CB1- and CB2-selective cannabinoid receptor antagonists are a new class of osteoclast inhibitors that may be of value in the treatment of osteoporosis and other bone diseases.