Targeted disruption of ephrin B1 in cells of myeloid lineage increases osteoclast differentiation and bone resorption in mice

Disruption of ephrin B1 in collagen I producing cells in mice results in severe skull defects and reduced bone formation. Because ephrin B1 is also expressed during osteoclast differentiation and because little is known on the role of ephrin B1 reverse signaling in bone resorption, we examined the bone phenotypes in ephrin B1 conditional knockout mice, and studied the function of ephrin B1 reverse signaling on osteoclast differentiation and resorptive activity. Targeted deletion of ephrin B1 gene in myeloid lineage cells resulted in reduced trabecular bone volume, trabecular number and trabecular thickness caused by increased TRAP positive osteoclasts and bone resorption. Histomorphometric analyses found bone formation parameters were not changed in ephrin B1 knockout mice. Treatment of wild-type precursors with clustered soluble EphB2-Fc inhibited RANKL induced formation of multinucleated osteoclasts, and bone resorption pits. The same treatment of ephrin B1 deficient precursors had little effect on osteoclast differentiation and pit formation. Similarly, activation of ephrin B1 reverse signaling by EphB2-Fc treatment led to inhibition of TRAP, cathepsin K and NFATc1 mRNA expression in osteoclasts derived from wild-type mice but not conditional knockout mice. Immunoprecipitation with NHERF1 antibody revealed ephrin B1 interacted with NHERF1 in differentiated osteoclasts. Treatment of osteoclasts with exogenous EphB2-Fc resulted in reduced phosphorylation of ezrin/radixin/moesin. We conclude that myeloid lineage produced ephrin B1 is a negative regulator of bone resorption in vivo, and that activation of ephrin B1 reverse signaling inhibits osteoclast differentiation in vitro in part via a mechanism that involves inhibition of NFATc1 expression and modulation of phosphorylation status of ezrin/radixin/moesin.     

The cytoskeletal regulatory scaffold protein GIT2 modulates mesenchymal stem cell differentiation and osteoblastogenesis

G protein-coupled receptor kinase interacting protein 2 (GIT2) is a signaling scaffold protein involved in the regulation of cytoskeletal structure, membrane trafficking, and G protein-coupled receptor internalization. Since dynamic cytoskeletal reorganization plays key roles both in osteoblast differentiation and in the maintenance of osteoclast polarity during bone resorption, we hypothesized that skeletal physiology would be altered in GIT2(-/-) mice. We found that adult GIT2(-/-) mice have decreased bone mineral density and bone volume in both the trabecular and cortical compartments. This osteopenia was associated with decreased numbers of mature osteoblasts, diminished osteoblastic activity, and increased marrow adiposity, suggesting a defect in osteoblast maturation. In vitro, mesenchymal stem cells derived from GIT2(-/-) mice exhibited impaired differentiation into osteoblasts and increased adipocyte differentiation, consistent with a role for GIT2 in mesenchymal stem cell fate determination. Despite elevated osteoclast inducing cytokines and osteoclast numbers, GIT2(-/-) mice also exhibit impaired bone resorption, consistent with a further role for GIT2 in regulating osteoclast function. Collectively, these findings underscore the importance of the cytoskeleton in both osteoblast and osteoclast function and demonstrate that GIT2 plays essential roles in skeletal metabolism, affecting both bone formation and bone resorption in vivo.     

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.     

Osteoprotegerin inhibit osteoclast differentiation and bone resorption by enhancing autophagy via AMPK/mTOR/p70S6K signaling pathway in vitro

Osteoclasts are highly differentiated terminal cells formed by fusion of hematopoietic stem cells. Previously, osteoprotegerin (OPG) inhibit osteoclast differentiation and bone resorption by blocking receptor activator of nuclear factor-κB ligand (RANKL) binding to RANK indirect mechanism. Furthermore, autophagy plays an important role during osteoclast differentiation and function. However, whether autophagy is involved in OPG-inhibited osteoclast formation and bone resorption is not known. To elucidate the role of autophagy in OPG-inhibited osteoclast differentiation and bone resorption, we used primary osteoclast derived from mice bone marrow monocytes/macrophages (BMM) by induced M-CSF and RANKL. The results showed that autophagy-related proteins expression were upregulated; tartrate-resistant acid phosphatase-positive osteoclast number and bone resorption activity were decreased; LC3 puncta and autophagosomes number were increased and activated AMPK/mTOR/p70S6K signaling pathway. In addition, chloroquine (as the autophagy/lysosome inhibitor, CQ) or rapamycin (as the autophagy/lysosome inhibitor, Rap) attenuated osteoclast differentiation and bone resorption activity by OPG treatment via AMPK/mTOR/p70S6K signaling pathway. Our data demonstrated that autophagy plays a critical role in OPG inhibiting osteoclast differentiation and bone resorption via AMPK/mTOR/p70S6K signaling pathway in vitro.     

Amylin inhibits bone resorption while the calcitonin receptor controls bone formation in vivo

Amylin is a member of the calcitonin family of hormones cosecreted with insulin by pancreatic beta cells. Cell culture assays suggest that amylin could affect bone formation and bone resorption, this latter function after its binding to the calcitonin receptor (CALCR). Here we show that Amylin inactivation leads to a low bone mass due to an increase in bone resorption, whereas bone formation is unaffected. In vitro, amylin inhibits fusion of mononucleated osteoclast precursors into multinucleated osteoclasts in an ERK1/2-dependent manner. Although Amylin +/- mice like Amylin-deficient mice display a low bone mass phenotype and increased bone resorption, Calcr +/- mice display a high bone mass due to an increase in bone formation. Moreover, compound heterozygote mice for Calcr and Amylin inactivation displayed bone abnormalities observed in both Calcr +/- and Amylin +/- mice, thereby ruling out that amylin uses CALCR to inhibit osteoclastogenesis in vivo. Thus, amylin is a physiological regulator of bone resorption that acts through an unidentified receptor.     

Effect of CD44 deficiency on in vitro and in vivo osteoclast formation

In vitro studies have shown that CD44 is involved in the fusion process of osteoclast precursor cells. Yet, in vivo studies do not support this, since an osteopetrotic phenotype has not been described for CD44 knock-out (CD44 k.o.) mice. This discrepancy may suggest that the role of CD44 in fusion may depend on the microenvironment of osteoclast formation. We investigated osteoclast formation of CD44 k.o. and wild-type mice under three conditions: in vitro, both on plastic and on bone and in vivo by analyzing osteoclast number, and size in long bones from wild-type and CD44 k.o. mice. Bone marrow cells from wild-type and CD44 k.o. mice were analyzed for their capacity to form osteoclasts on plastic and on bone in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of NF-kB ligand (RANKL). On plastic, the number of multinucleated tartrate resistant acid phosphatase (TRAP) positive cells in CD44 k.o. cultures was twofold higher than in wild-type cultures. On bone, however, equal numbers of osteoclasts were formed. Interestingly, the total number of osteoclasts formed on bone proved to be higher than on plastic for both genotypes, strongly suggesting that osteoclastogenesis was stimulated by the bone surface, and that CD44 is not required for osteoclast formation on bone. Functional analyses showed that bone resorption was similar for both genotypes. We further studied the osteoclastogenic potential of wild-type bone marrow cells in the presence of CD44 blocking antibodies. Osteoclastogenesis was not affected by these antibodies, a further indication that CD44 is not required for the formation of multinucleated cells. Finally, we analyzed the in vivo formation of osteoclasts by analyzing long bones from wild-type and CD44 k.o. mice. Morphometric analysis revealed no difference in osteoclast number, nor in number of nuclei per osteoclasts or in osteoclast size. Our in vitro experiments on plastic showed an enhanced formation of osteoclasts in the absence of CD44, thus suggesting that CD44 has an inhibitory effect on osteoclastogenesis. However, when osteoclasts were generated on bone, no differences in number of multinucleated cells nor in bone resorption were seen. These observations are in agreement with in vivo osteoclast characteristics, where no differences between wild-type and CD44 k.o. bones were encountered. Therefore, the modulating role of CD44 in osteoclast formation appears to depend on the microenvironment.     

The possible role of TGF-beta-induced suppressors of cytokine signaling expression in osteoclast/macrophage lineage commitment in vitro

Osteoclast formation is dependent on the ability of TGF-beta to enable receptor activator of NF-kappaB ligand (RANKL)-induced commitment of hemopoietic precursors to the osteoclastic lineage. The mechanism by which TGF-beta enables formation is unknown. One possibility is that TGF-beta opposes Janus kinase (JAK)/STAT signals generated by inhibitory cytokines such as IFN-beta. The JAK/STAT pathway is activated by cytokines that induce resistance to osteoclast formation, such as IFN-gamma and M-CSF, and the effect of these is opposed by TGF-beta. Recently, a group of STAT-induced factors, termed suppressors of cytokine signaling (SOCS), has been identified that inhibit JAK/STAT signals. Therefore, we tested the ability of TGF-beta to induce SOCS expression in osteoclast precursors and examined the effect of SOCS expression on osteoclast/macrophage lineage commitment. We found that while SOCS mRNA is undetectable in macrophages, osteoclasts express SOCS-3, and TGF-beta up-regulates this expression. Furthermore, TGF-beta rapidly induces sustained SOCS-3 expression in macrophage/osteoclast precursors. To determine whether SOCS-3 plays a role in osteoclast differentiation we expressed SOCS-3 in precursors using a retroviral system. We found that osteoclast differentiation was significantly enhanced in SOCS-3-infected precursors, and SOCS-3 expression enables formation in the presence of anti-TGF-beta Ab. On the other hand, antisense knockdown of SOCS-3 strongly suppressed osteoclast formation and significantly blunted the response to TGF-beta. Moreover, like TGF-beta, SOCS-3 expression opposed the inhibitory effect of IFN-beta. These data suggest that TGF-beta-induced expression of SOCS-3 may represent a mechanism by which TGF-beta suppresses inhibitory cytokine signaling, priming precursors for a role in bone resorption.     

Osteoclast inhibitory lectin, an immune cell product that is required for normal bone physiology in vivo

Osteoclast inhibitory lectin (OCIL or clrb) is a member of the natural killer cell C-type lectins that have a described role mostly in autoimmune cell function. OCIL was originally identified as an osteoblast-derived inhibitor of osteoclast formation in vitro. To determine the physiological function(s) of OCIL, we generated ocil(-/-) mice. These mice appeared healthy and were fertile, with no apparent immune function defect, and phenotypic abnormalities were limited to bone. Histomorphometric analysis revealed a significantly lower tibial trabecular bone volume and trabecular number in the 10- and 16-week-old male ocil(-/-) mice compared with wild type mice. Furthermore, ocil(-/-) mice showed reduced bone formation rate in the 10-week-old females and 16-week-old males while Static markers of bone formation showed no significant changes in male or female ocil(-/-) mice. Examination of bone resorption markers in the long bones of ocil(-/-) mice indicated a transient increase in osteoclast number per unit bone perimeter. Enhanced osteoclast formation was also observed when either bone marrow or splenic cultures were generated in vitro from ocil(-/-) mice relative to wild type control cultures. Loss of ocil therefore resulted in osteopenia in adult mice primarily as a result of increased osteoclast formation and/or decreased bone formation. The enhanced osteoclastic activity led to elevated serum calcium levels, which resulted in the suppression of circulating parathyroid hormone in 10-week-old ocil(-/-) mice compared with wild type control mice. Collectively, our data suggest that OCIL is a physiological negative regulator of bone.     

Expression of typical osteoclast markers by PBMCs after PEG-induced fusion as a model for studying osteoclast differentiation

Bone is a metabolically active organ subjected to continuous remodeling process that involves resorption by osteoclast and subsequent formation by osteoblasts. Osteoclast involvement in this physiological event is regulated by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL). Fusion of mono-nuclear pre-osteoclasts is a critical event for osteoclast differentiation and for bone resorption. Here we show that PBMCs can be successfully fused with polyethylenglicol (PEG) in order to generated viable osteoclast-like cells that exhibit tartrate-resistant acid phosphatase (TRAP) and bone resorptive activities. PEG-fused PBMCs expressed additional markers compatible with osteoclastogenic differentiation such as carbonic anhydrase II (CAII), calcitonin receptor (CR), cathepsin K (Cat K), vacuolar ATPase (V-ATPase) subunit C1 (V-ATPase), integrin β3, RANK and cell surface aminopeptidase N/CD13. Actin redistribution in PEG-fused cells was found to be affected by cell cycle synchronization at G0/G1 or G2/M phases. PEG-induced fusion also led to expression of tyrosine kinases c-Src and Syk in their phosphorylated state. Scanning electron microscopy images showed morphological features typical of osteoclast-like cells. The results here shown allow concluding that PEG-induced fusion of PBMCs provides a suitable model system for understanding the mechanisms involved in osteoclastogenesis and for assaying new therapeutic strategies.     

The roles of prostanoids, leukotrienes, and platelet-activating factor in bone metabolism and disease

The production of a variety of lipid mediators is enhanced in bone-resorptive diseases such as osteoporosis, rheumatoid arthritis, osteoarthritis, and periodontitis. Prostaglandin E(2) (PGE(2)) is one of the most notable lipid mediators of bone remodeling, and has been linked clinically to many bone-resorptive diseases. In vitro studies with bone cell cultures have demonstrated that the bone-resorptive activity of PGE(2), which is mediated by receptor activator of NF-kappaB ligand (RANKL), is key for the induction of osteoclast formation. Furthermore, interleukin (IL)-1- and IL-6-stimulated bone resorption involves PGE(2) production. In addition to its bone-resorptive effects, PGE(2) promotes bone formation in vitro by stimulating osteoblastic proliferation and differentiation. The multifaceted nature of PGE(2) makes it difficult to discern its role during bone remodeling. Leukotrienes (LTs), and particularly LTB(4), have also been implicated in bone remodeling and disease-specifically in rheumatoid arthritis. Moreover, recent studies from our laboratory have shown that platelet-activating factor (PAF) receptor-deficient mice develop only mild osteoporosis. Osteoclast survival in these mice is shortened and osteoclastic bone resorption is impaired. This review article focuses on these families of lipids and their function during bone metabolism and disease.