Osteoclast differentiation and function in aquaglyceroporin AQP9-null mice

Background information. Osteoclasts are cells specialized for bone resorption and play important roles in bone growth and calcium homoeostasis. Differentiation of osteoclasts involves fusion of bone marrow macrophage mononuclear precursors in response to extracellular signals. A dramatic increase in osteoclast cell volume occurs during osteoclast biogenesis and is believed to be mediated by AQP9 (aquaporin 9), a membrane protein that can rapidly transport water and other small neutral solutes across cell membranes. Results. In the present study we report an increase in expression of AQP9 during differentiation of a mouse macrophage cell line into osteoclasts. Bone marrow macrophages from wild‐type and AQP9‐null mice differentiate into osteoclasts that have similar morphology, contain comparable numbers of nuclei, and digest synthetic bone to the same extent. Bones from wild‐type and AQP9‐null mice contain similar numbers of osteoclasts and have comparable density and structure as measured by X‐ray absorptiometry and microcomputed tomography. Conclusions. Our results confirm that AQP9 expression rises during osteoclast biogenesis, but indicate that AQP9 is not essential for osteoclast function or differentiation under normal physiological conditions.     

Osteoclast differentiation factor acts as a multifunctional regulator in murine osteoclast differentiation and function.

Osteoclast differentiation factor (ODF), a novel member of the TNF ligand family, is expressed as a membrane-associated protein by osteoblasts/stromal cells. The soluble form of ODF (sODF) induces the differentiation of osteoclast precursors into osteoclasts in the presence of M-CSF. Here, the effects of sODF on the survival, multinucleation, and pit-forming activity of murine osteoclasts were examined in comparison with those of M-CSF and IL-1. Osteoclast-like cells (OCLs) formed in cocultures of murine osteoblasts and bone marrow cells expressed mRNA of RANK (receptor activator of NF-kappaB), a receptor of ODF. The survival of OCLs was enhanced by the addition of each of sODF, M-CSF, and IL-1. sODF, as well as IL-1, activated NF-kappaB and c-Jun N-terminal protein kinase (JNK) in OCLs. Like M-CSF and IL-1, sODF stimulated the survival and multinucleation of prefusion osteoclasts (pOCs) isolated from the coculture. When pOCs were cultured on dentine slices, resorption pits were formed on the slices in the presence of either sODF or IL-1 but not in that of M-CSF. A soluble form of RANK as well as osteoprotegerin/osteoclastogenesis inhibitory factor, a decoy receptor of ODF, blocked OCL formation and prevented the survival, multinucleation, and pit-forming activity of pOCs induced by sODF. These results suggest that ODF regulates not only osteoclast differentiation but also osteoclast function in mice through the receptor RANK.     

Osteoclast activity modulates B-cell development in the bone marrow

B-cell development is dependent on the interactions between B-cell precursors and bone marrow stromal cells, but the role of osteoclasts (OCLs) in this process remains unknown. B lymphocytopenia is a characteristic of osteopetrosis, suggesting a modulation of B lymphopoiesis by OCL activity. To address this question, we first rescued OCL function in osteopetrotic oc/oc mice by dendritic cell transfer, leading to a restoration of both bone phenotype and B-cell development. To further explore the link between OCL activity and B lymphopoiesis, we induced osteopetrosis in normal mice by injections of zoledronic acid (ZA), an inhibitor of bone resorption. B-cell number decreased specifically in the bone marrow of ZA-treated mice. ZA did not directly affect B-cell differentiation, proliferation and apoptosis, but induced a decrease in the expression of CXCL12 and IL-7 by stromal cells, associated with reduced osteoblastic engagement. Equivalent low osteoblastic engagement in oc/oc mice confirmed that it resulted from the reduced OCL activity rather than from a direct effect of ZA on osteoblasts. These dramatic alterations of the bone microenvironment were disadvantageous for B lymphopoiesis, leading to retention of B-cell progenitors outside of their bone marrow niches in the ZA-induced osteopetrotic model. Altogether, our data revealed that OCLs modulate B-cell development in the bone marrow by controlling the bone microenvironment and the fate of osteoblasts. They provide novel basis for the regulation of the retention of B cells in their niche by OCL activity.     

Osteoclast lineage and function

Osteoclasts are members of the monocyte/macrophage lineage and are formed by cellular fusions from their mononuclear precursors. Their differentiation is regulated by a number of other cells and their products, especially by RANKL and M-CSF. The resorbing osteoclasts are polarized and show specific plasma membrane domains. Polarization and bone resorption need a continuous membrane trafficking and modulation of the cytoskeleton. The most characteristic feature of osteoclasts is their unique capacity to dissolve crystalline hydroxyapatite by targeted secretion of HCl into the extracellular resorption lacuna. Organic matrix is degraded by enzymes like cathepsin K and the degradation products are transcytosed through the cell for secretion. Dissolution of hydroxyapatite releases large amounts of soluble calcium, phosphate and bicarbonate. Removal of these ions apparently involves the vesicular pathways and direct ion transport via different ion exchangers, channels and pumps. Detailed molecular knowledge of osteoclast differentiation and function has helped us to identify several target molecules and develop specific treatments to inhibit pathological bone resorption in various skeletal diseases.     

Osteoclast radicals

In biological research, new ideas arise and quickly spread to encompass the entire field. Thus, the evolution of molecular biology has significantly changed our methods of approaching our research. A similar far-reaching finding has been the advent of radical reactions into biology. Although radical chemistry has been utilzed for many technological advances that affect our daily lives, the appreciation of this same process within our cells has opened an unexplored arena for research enquiry. As cellular messengers, radical molecules seem shimsically designed: they are evanescent, rapidly and apparently indiscriminately reactive, and barely detectable bymost biological methods. Yet, our initial probing of these reactive agents in cells and organisms has led us to postulate a virtually undescribed system of communication within and among cells which may have significant effects in multiple organs. In bone, radical reactions have been attributed with an important role in the control of bone resorption.     

Osteoclast receptors and signaling

Osteoclasts are bone-resorbing cells derived from hematopoietic precursors of the monocyte-macrophage lineage. Besides the well known Receptor Activator of Nuclear factor-κB (RANK), RANK ligand and osteoprotegerin axis, a variety of factors tightly regulate osteoclast formation, adhesion, polarization, motility, resorbing activity and life span, maintaining bone resorption within physiological ranges. Receptor-mediated osteoclast regulation is rather complex. Nuclear receptors, cell surface receptors, integrin receptors and cell death receptors work together to control osteoclast activity and prevent both reduced or increased bone resorption. Here we will discuss the signal transduction pathways activated by the main osteoclast receptors, integrating their function and mechanisms of action.     

Osteoclast differentiation by human osteoblastic cell line SaOS-2 primed with bacterial lipid A

We examined the responses of human osteoblastic cell line SaOS-2 to bacterial lipid A, a bioactive center of lipopolysaccharide, during osteoclast differentiation of human peripheral blood mononuclear cells (PBMC). SaOS-2 cells expressed mRNA for Toll-like receptor (TLR) 4, MD-2, CD14, and myeloid differentiation factor 88, whereas they failed to express mRNA for TLR2. Escherichia coli-type synthetic lipid A (compound 506) induced cytokine mRNA expression and nuclear factor (NF)-kappaB activation in SaOS-2 cells. Compound 506 also increased the expression of receptor activator of NF-kappaB ligand. Further, cells primed with compound 506 augmented the differentiation of PBMC into osteoclastic cells, and the effect was inhibited by anti-TLR4 monoclonal antibody. These findings suggest that the TLR signaling cascade in osteoblastic cells is involved in regulating the function of osteoclastogenesis.     

Osteoclast differentiation is impaired in the absence of inhibitor of kappa B kinase alpha

Signaling through the receptor activator of nuclear factor kappa B (RANK) is required for both osteoclast differentiation and mammary gland development, yet the extent to which RANK utilizes similar signaling pathways in these tissues remains unclear. Mice expressing a kinase-inactive form of the inhibitor of kappa B kinase alpha (IKK alpha) have mammary gland defects similar to those of RANK-null mice yet have apparently normal osteoclast function. Because mice that completely lack IKK alpha have severe skin and skeletal defects that are not associated with IKK alpha-kinase activity, we wished to directly examine osteoclastogenesis in IKK alpha(-/-) mice. We found that unlike RANK-null mice, which completely lack osteoclasts, IKK alpha(-/-) mice did possess normal numbers of TRAP(+) osteoclasts. However, only 32% of these cells were multinucleated compared with 57% in wild-type littermates. A more profound defect in osteoclastogenesis was observed in vitro using IKK alpha(-/-) hematopoietic cells treated with colony-stimulating factor 1 and RANK ligand (RANKL), as the cells failed to form large, multinucleated osteoclasts. Additionally, overall RANKL-induced global gene expression was significantly blunted in IKK alpha(-/-) cells, including osteoclast-specific genes such as TRAP, MMP-9, and c-Src. IKK alpha was not required for RANKL-mediated I kappa B alpha degradation or phosphorylation of mitogen-activated protein kinases but was required for RANKL-induced p100 processing. Treatment of IKK alpha(-/-) cells with tumor necrosis factor alpha (TNF alpha) in combination with RANKL led to partial rescue of osteoclastogenesis despite a lack of p100 processing. However, the ability of TNF alpha alone or in combination with transforming growth factor beta to induce osteoclast differentiation was dependent on IKK alpha, suggesting that synergy between RANKL and TNFalpha can overcome p100 processing defects in IKK alpha(-/-) cells.     

Interleukin-32 Promotes Osteoclast Differentiation but Not Osteoclast Activation

Background

Interleukin-32 (IL-32) is a newly described cytokine produced after stimulation by IL-2 or IL-18 and IFN-γ. IL-32 has the typical properties of a pro-inflammatory mediator and although its role in rheumatoid arthritis has been recently reported its effect on the osteoclastogenesis process remains unclear.

Methodology/Principal Findings

In the present study, we have shown that IL-32 was a potent modulator of osteoclastogenesis in vitro, whereby it promoted the differentiation of osteoclast precursors into TRAcP+ VNR+ multinucleated cells expressing specific osteoclast markers (up-regulation of NFATc1, OSCAR, Cathepsin K), but it was incapable of inducing the maturation of these multinucleated cells into bone-resorbing cells. The lack of bone resorption in IL-32-treated cultures could in part be explain by the lack of F-actin ring formation by the multinucleated cells generated. Moreover, when IL-32 was added to PBMC cultures maintained with soluble RANKL, although the number of newly generated osteoclast was increased, a significant decrease of the percentage of lacunar resorption was evident suggesting a possible inhibitory effect of this cytokine on osteoclast activation. To determine the mechanism by which IL-32 induces such response, we sought to determine the intracellular pathways activated and the release of soluble mediators in response to IL-32. Our results indicated that compared to RANKL, IL-32 induced a massive activation of ERK1/2 and Akt. Moreover, IL-32 was also capable of stimulating the release of IL-4 and IFN-γ, two known inhibitors of osteoclast formation and activation.

Conclusions/Significance

This is the first in vitro report on the complex role of IL-32 on osteoclast precursors. Further clarification on the exact role of IL-32 in vivo is required prior to the development of any potential therapeutic approach.     

Osteoclast signalling pathways

The osteoclast is a monocyte-derived cell with complex regulatory control due to its role, balancing calcium homeostasis with skeletal modelling and repair. Normal differentiation requires tyrosine kinase- and tumor necrosis-family receptors, normally fms and RANK. Ligands for these receptors plus unidentified serum or cell-presented factor(s) are needed for in vitro differentiation, possibly signalling via an immune-like tyrosine kinase acceptor molecule. Osteoclast development and activity are increased by cytokines signalling through GP130, such as IL-6, by TGF-beta, and by IL-1, although these cannot replace serum. Other tyrosine kinase receptors including kit and met can augment fms signalling, and TNFs other than RANKL, including TNFalpha and TRAIL, modify RANK signalling, which is also susceptible to interference by interferons. The situation is further complicated by G-protein coupled receptors including the calcitonin receptor, by integrin or calcium-mediated signals, and by estrogen receptors, which operate in bone largely via NO downstream signals. Differentiation, activity, and survival signals merge in intracellular second messengers. These include cytoplasmic kinases of several families; differentiation pathways often terminate in Erk/Jun kinases or NF-kappaB. Key regulatory intermediates include TRAF6, src, Smad3, phosphatidylinositol-3-kinase, Jak/Stat, and the cGMP-dependent protein kinase I. There are substantial uncertainties regarding how intracellular agents connect to primary signals. The frontier includes characterization of how scaffolding/adapter proteins, such as cbl, gab, grb, p130Cas, and shc, as well as itam-containing proteins and nonreceptor tyrosine kinase adapters of the src and syk families, delimit and integrate signals of multiple receptors to bring about specific outcomes.     

Histone differentiation and nuclear activity

When fast green and eosin are used in combination to stain histones, nuclei display different affinities toward the dyes, some binding fast green exclusively, others binding eosin exclusively, and still others, both stains. In a given tissue, the frequencies of nuclei exhibiting the different colors remain fairly constant over a wide range of staining conditions. Nuclei of cells of the same type may stain differently, but when they are in the same stage of development or state of activity they tend to stain alike. Xenopus erythrocyte nuclei stain bright pink. Condensed mitotic and meiotic chromosomes stain purple. In the grasshopper spermatocyte, the main body of the interphase nucleus stains bright green, but the condensed chromosome stains purple. The mole crab sperm contains several distinct histone-like proteins, that differ in their amino acid compositions, within separate areas of the cell. In these sperms, the lysine-rich histones bind eosin, while the protamine-like protein and arginine-rich histone bind fast green. In general, the eosin and fast green bind preferentially to the lysine and arginine rich histones respectively, when the dyes are permitted to compete with one another. In several systems, including spermiogenesis and erythropoiesis, the aquisition of an eosinophilic component by the nuclei accompanies the slowing of RNA synthesis, and it is suggested that there may be a causal relationship between the two events, the eosinophilic histone effecting RNA synthesis within the nucleus as a whole.     

Vinculin Regulates Osteoclast Function

Osteoclastic bone resorption depends upon the cell''s ability to organize its cytoskeleton. Because vinculin (VCL) is an actin-binding protein, we asked whether it participates in skeletal degradation. Thus, we mated VCL^fl/fl mice with those expressing cathepsin K-Cre (CtsK-VCL) to delete the gene in mature osteoclasts or lysozyme M-Cre (LysM-VCL) to target all osteoclast lineage cells. VCL-deficient osteoclasts differentiate normally but, reflecting cytoskeletal disorganization, form small actin rings and fail to effectively resorb bone. In keeping with inhibited resorptive function, CtsK-VCL and LysM-VCL mice exhibit a doubling of bone mass. Despite cytoskeletal disorganization, the capacity of VCL^−/− osteoclastic cells to normally phosphorylate c-Src in response to αvβ3 integrin ligand is intact. Thus, integrin-activated signals are unrelated to the means by which VCL organizes the osteoclast cytoskeleton. WT VCL completely rescues actin ring formation and bone resorption, as does VCL^P878A, which is incapable of interacting with Arp2/3. As expected, deletion of the VCL tail domain (VCL^1–880), which binds actin, does not normalize VCL^−/− osteoclasts. The same is true regarding VCL^I997A, which also prevents VCL/actin binding, and VCL^A50I and VCL^811–1066, both of which arrest talin association. Thus, VCL binding talin, but not Arp2/3, is critical for osteoclast function, and its selective inhibition retards physiological bone loss.     

Osteoclast resorption-stimulating activity is associated with the osteoblast cell surface and/or the extracellular matrix

Osteoblasts mediate much of the hormonal responsiveness of osteoclasts. We and others have found that one mechanism through which this regulation is effected is by release of osteoclast resorption-stimulating activity (ORSA) into culture supernatants. However, although hormonal responsiveness is regularly observed in co-cultures, ORSA is not always detectable in conditioned media. We show here that one explanation for this finding is that ORSA may be retained by heparin-like glycosaminoglycans (GAGs) of the cell surface or extracellular matrix of osteoblasts. We found that protease-sensitive ORSA could be extracted from monolayers of the osteoblastic cell line UMR 106 with 2M NaCl or collagenase. Production of this activity was increased in response to 1,25(OH)2D3. The presence of the GAG heparin was required to reveal ORSA. Immobilisation of ORSA by GAGs may assist osteoblastic cells in the regulation of the complex patterns of osteoclastic activity observed during skeletal morphogenesis and restructuring.     

Osteoclast differentiation factor modulates cell cycle machinery and causes a delay in s phase progression in RAW264 cells

Osteoclast differentiation factor (ODF) induces differentiation of mouse RAW264 cells to mature osteoclasts. To understand the mechanism controlling a coupling between withdrawal from the cell cycle and differentiation, we examined cell cycle progression and expression profiles of cell cycle regulatory genes at the initial phase in committed cells. ODF rapidly converted the hyperphosphorylated form of the retinoblastoma protein (pRb) into the hypophosphorylated form. The p21 protein was induced by ODF treatment in the same time course with that of dephosphorylation of pRb, followed by a sharp decline. After this period, a delayed entry of the S phase started accompanying the induction of CycD3 and cdk6 in differentiating cells. Hydroxyurea treatment indicated that the S phase entry was a prerequisite for osteoclast formation. Thus, ODF induces pleiotropic effects on cell cycle regulatory genes in RAW264 cells during the initial phase of the differentiation process to osteoclasts.     

Phosphatidylinositol 3-Kinase Association with the Osteoclast Cytoskeleton, and Its Involvement in Osteoclast Attachment and Spreading

Osteoclast activation involves attachment to the mineralized bone matrix and reorganization of the cytoskeleton, leading to polarization of the cell. Signaling molecules, PI3-kinase, rho A, and pp60^c-src, were shown to be essential for osteoclastic bone resorption. In this study we have focused on the involvement of these signaling molecules in the early event of osteoclast activation: attachment, spreading, and organization of the cytoskeleton. Highly purified osteoclasts were fractionated into Triton X-100-soluble or cytosolic and Triton X-100-insoluble or cytoskeletal fractions, and the distribution of above-mentioned signaling molecules between the two fractions was examined. PI3-kinase, rho A, and pp60^c-srcall showed translocation to the cytoskeletal fraction upon osteoclast attachment to plastic. However, PI3-kinase and rho A, but not pp60^c-src, showed further translocation of 2.4- and 3.2-fold, respectively, upon attachment of osteoclasts to bone. PI3-kinase translocation to the cytoskeleton was inhibited by either cytochalasin B or colchicine. Furthermore, treatment of osteoclasts with the PI3-kinase inhibitor wortmannin decreased its translocation, suggesting that PI3-kinase activity was needed for its translocation. Moreover, wortmannin inhibited osteoclast attachment to both bone and plastic and caused drastic changes in osteoclast morphology resulting in rounding of the cells, disappearance of F-actin structures or podosomes, and appearance of punctate or vesicular structures inside the cells. Osteoblastic MB1.8 cells and IC-21 macrophages did not show additional translocation of PI3-kinase or rho A upon attachment to bone or changes in attachment or morphology in response to wortmannin. Finally, PI3-kinase coimmunoprecipitated with α_vβ₃integrin from osteoclasts.     

Transglutaminase activity and embryonal carcinoma cell differentiation

Murine embryonal carcinoma (EC) cells induced to differentiate by retinoic acid (RA) modulate transglutaminase (TGase) activity shortly after exposure to the inducer. Compounds that inhibit TGase enzyme activity in vitro can successfully block RA induced EC cell differentiation in culture. These observations suggest that TGase may play a role in mediating RA induced EC cell differentiation.