Giant cell tumor of bone: A unique paradigm of stromal–hematopoietic cellular interactions

Giant cell tumor of bone is a progressive, potentially malignant process which destroys skeletal tissue by virtue of its osteoclast complement. As a biological entity it provides a unique natrual model of bone resorption by osteoclasts whose recruitment and development is controlled by a neoplastic population of fibroblast-like cells. Understanding of the etiopathogenesis of this tumor could provide new insights into the mechanisms underlying osteoblast–osteoclast interactions in normal and diseased bone. Recent studies have shown that the stromal cell component in giant cell tumors is the only proliferating subpopulation of cells, and the giant cells themselves are nonproliferative and reactive. These stromal cells express several genes associated with the osteoblastic phenotype, synthesize, to a limited degree, certain matrix proteins associated with bone, and express several factors which are presumably involved in the recruitment of osteoclasts. In culture, giant cell tumor–associated stromal cells promote the fusion of monocytes and the proliferation of osteoblasts either by the secretion of factors or cell–cell contact. Hence, giant cell tumor of bone is a self-contained biosystem in which cells of both the stromal and hematopoietic lineages interact in a fashion similar to that observed in normal skeletal remodeling. The neoplastic nature of the stromal component, however, drives the hematopoietic precursors to undergo fusion, produces aggressive bone resorption, and results in extensive skeletal destruction. Examination of the various components of this system could lead to new directions for investigations aimed at a better understanding of osteoblast–osteoclast interactions. © 1994 Wiley-Liss, Inc.     

Osteoclast-derived activity in the coupling of bone formation to resorption

The cells of bone and the immune system communicate by means of soluble and membrane-bound cytokines and growth factors. Through local signalling mechanisms, cells of the osteoblast lineage control the formation and activity of osteoclasts and, therefore, the resorption of bone. Both T and B lymphocytes produce activators and inhibitors of osteoclast formation. A local 'coupling factor' linking bone resorption to subsequent formation in the bone multicellular unit (BMU) has long been proposed as the key regulator of the bone remodelling process, but never identified. There is evidence in support of the view that the coupling mechanism is dependent on growth factors released from the bone matrix during resorption, or is generated from maturing osteoblasts. We argue that osteoclasts contribute in important ways to the transiently activated osteoclast, and stimulate osteoblast lineage cells to begin replacing the resorbed bone in each BMU.     

Osteoclast-osteoblast communication

Cells in osteoclast and osteoblast lineages communicate with each other through cell-cell contact, diffusible paracrine factors and cell-bone matrix interaction. Osteoclast-osteoblast communication occurs in a basic multicellular unit (BMU) at the initiation, transition and termination phases of bone remodeling. At the initiation phase, hematopoietic precursors are recruited to the BMU. These precursors express cell surface receptors including c-Fms, RANK and costimulatory molecules, such as osteoclast-associated receptor (OSCAR), and differentiate into osteoclasts following cell-cell contact with osteoblasts, which express ligands. Subsequently, the transition from bone resorption to formation is mediated by osteoclast-derived ‘coupling factors’, which direct the differentiation and activation of osteoblasts in resorbed lacunae to refill it with new bone. Bidirectional signaling generated by interaction between ephrinB2 on osteoclasts and EphB4 on osteoblast precursors facilitates the transition. Such interaction is likely to occur between osteoclasts and lining cells in the bone remodeling compartment (BRC). At the termination phase, bone remodeling is completed by osteoblastic bone formation and mineralization of bone matrix. Here, we describe molecular communication between osteoclasts and osteoblasts at distinct phases of bone remodeling.     

Cadherin-6 Mediates the Heterotypic Interactions between the Hemopoietic Osteoclast Cell Lineage and Stromal Cells in a Murine Model of Osteoclast Differentiation

Osteoclasts are multinucleated cells of hemopoietic origin that are responsible for bone resorption during physiological bone remodeling and in a variety of bone diseases. Osteoclast development requires direct heterotypic cell–cell interactions of the hemopoietic osteoclast precursors with the neighboring osteoblast/stromal cells. However, the molecular mechanisms underlying these heterotypic interactions are poorly understood. We isolated cadherin-6 isoform, denoted cadherin-6/2 from a cDNA library of human osteoclast-like cells. The isolated cadherin-6/2 is 3,423 bp in size consisting of an open reading frame of 2,115 bp, which encodes 705 amino acids. This isoform lacks 85 amino acids between positions 333 and 418 and contains 9 different amino acids in the extracellular domain compared with the previously described cadherin-6. The human osteoclast-like cells also expressed another isoform denoted cadherin-6/1 together with the cadherin-6. Introduction of cadherin-6/2 into L-cells that showed no cell–cell contact caused evident morphological changes accompanied with tight cell–cell association, indicating the cadherin-6/2 we isolated here is functional. Moreover, expression of dominant-negative or antisense cadherin-6/2 construct in bone marrow–derived mouse stromal ST2 cells, which express only cadherin-6/2, markedly impaired their ability to support osteoclast formation in a mouse coculture model of osteoclastogenesis. Our results suggest that cadherin-6 may be a contributory molecule to the heterotypic interactions between the hemopoietic osteoclast cell lineage and osteoblast/bone marrow stromal cells required for the osteoclast differentiation. Since both osteoclasts and osteoblasts/bone marrow stromal cells are the primary cells controlling physiological bone remodeling, expression of cadherin-6 isoforms in these two cell types of different origin suggests a critical role of these molecules in the relationship of osteoclast precursors and cells of osteoblastic lineage within the bone microenvironment.     

TNAP, TrAP, ecto-purinergic signaling, and bone remodeling

Bone remodeling is a process of continuous resorption and formation/mineralization carried out by osteoclasts and osteoblasts, which, along with osteocytes, comprise the bone multicellular unit (BMU). A key component of the BMU is the bone remodeling compartment (BRC), isolated from the marrow by a canopy of osteoblast-like lining cells. Although much progress has been made regarding the cytokine-dependent and hormonal regulation of bone remodeling, less attention has been placed on the role of extracellular pH (pH(e)). Osteoclastic bone resorption occurs at acidic pH(e). Furthermore, osteoclasts can be regarded as epithelial-like cells, due to their polarized structure and ability to form a seal against bone, isolating the lacunar space. The major ecto-phosphatases of osteoclasts and osteoblasts, acid and alkaline phosphatases, both have ATPase activity with pH optima several units different from neutrality. Furthermore, osteoclasts and osteoblasts express plasma membrane purinergic P2 receptors that, upon activation by ATP, accelerate bone osteoclast resorption and impair osteoblast mineralization. We hypothesize that these ecto-phosphatases help regulate [ATP](e) and localized pH(e) at the sites of bone resorption and mineralization by pH-dependent ATP hydrolysis coupled with P2Y-dependent regulation of osteoclast and osteoblast function. Furthermore, osteoclast cellular HCO3(-), formed as a product of lacunar V-ATPase H(+) secretion, is secreted into the BRC, which could elevate BRC pH(e), in turn affecting osteoblast function. We will review the existing data addressing regulation of BRC pH(e), present a hypothesis regarding its regulation, and discuss the hypothesis in the context of the function of proteins that regulate pH(e).     

Effect of oestradiol on cytokine production in immortalized human marrow stromal cell lines.

Oestrogen deficiency enhances bone osteoclastogenesis and bone resorption. Evidence of cooperation between stromal cells and osteoclast precursors in mice suggests that oestradiol acts by regulating cytokine release from stromal cells. Bone marrow stroma contains multipotent progenitors that give rise to many mesenchymal lineages, including osteoblasts that may regulate osteoclast differentiation. We immortalized and characterized six human bone marrow stromal cell lines (presence of Stro1, secretion of alkaline phosphatase, osteocalcin, formation of lipid droplets, and presence of alpha and beta oestrogen receptors). The response of cytokines to oestradiol was then evaluated in vitro, as were the phorbol myristate acetate (PMA)-stimulated cytokine levels. Cells had the characteristics of undifferentiated stromal cells (Stro1+, RANK-L+), and expressed alpha-oestrogen receptors. The osteoblast phenotype (amounts of alkaline phosphatase and osteocalcin) was weak and there was a poor capacity to differentiate into adipocytes. These cell lines did not respond to oestradiol by producing interleukin 6 (IL-6), IL-1 or tumour necrosis factor alpha (TNF-alpha) either constitutively or after stimulation with PMA. Moreover, RANK-L and osteoprotegerin expressions were not regulated by oestradiol in vitro. Thus, modulation of these cytokines by stromal cells do not appear to be the mechanism by which oestradiol regulates bone resorption in humans.     

Osteoblastic regulation of osteoclast survival: effect of calcitriol

Throughout life, bone is remodelled in a dynamic process which results in a balance between bone formation by osteoblasts and bone resorption by osteoclasts. It is now clearly established that osteoblasts/stromal cells are crucial for differentiation of osteoclasts, through a mechanism involving cell-to-cell contact. However, the possible involvement of osteoblasts and stromal cells in the survival of osteoclasts has not yet been clearly demonstrated. In this study, we assessed the influence of cellular microenvironment, especially osteoblasts, on the osteoclast survival. Our results have shown significant differences in osteoclastic survival between unfractionated bone cells and pure osteoclasts. Furthermore, we have shown that addition of 1.25(OH)2D3 to unfractionated bone cells resulted in a dose-dependent increase in osteoclast survival. Finally, we have shown that a conditioned medium obtained from rat osteoblastic cells cultured with calcitriol was able to increase significantly survival of pure osteoclasts. Taken together, these results strongly suggest that osteoblastic cells present in the bone microenvironment might play a role in the osteoclastic survival by producing soluble factor which modulate osteoclast apoptosis.     

Osteoblast-osteoclast relationships in bone resorption: osteoblasts enhance osteoclast activity in a serum-free co-culture system.

Osteoblast-osteoclast relationships in bone resorption are unclear. We investigated whether osteoblasts constitutively influence osteoclast activity. We employed a serum-free co-culture system in which chicken osteoclasts and chick calvaria or, alternatively, isolated chick osteoblasts were cultured in two different compartments separated by a 0.45 micron porous membrane permeable to soluble molecules. Osteoclastic bone resorption, evaluated by release of 3H-proline from prelabeled bone fragments, was significantly enhanced by bone cells resident in the calvaria, as well as by isolated osteoblasts. Stimulation was specific, since periosteal cells, or skin fibroblasts, failed to mimic osteoblast activity. Conditioned medium from osteoblast cultures stimulated osteoclast function in a similar manner, indicating that paracrine signals, capable of crossing the porous membrane separating the two compartments, are released by the bone forming cells.     

CELLULAR BIOLOGY OF BONE RESORPTION

Past knowledge and the recent developments on the formation, activation and mode of action of osteoclasts, with particular reference to the regulation of each individual step, have been reviewed. The following conclusions of consensus have emerged.
1. The resorption of bone is the result of successive steps that can be regulated individually.
2. Osteoclast progenitors are formed in bone marrow. This is followed by their vascular dissemination and the generation of resting preosteoclasts and osteoclasts in bone.
3. The exact pathways of differentiation of the osteoclast progenators to mature osteoclasts are debatable, but there is clear evidence that stromal cells support osteoclast generation.
4. Osteoclasts are activated following contact with mineralized bone. This appears to be controlled by osteoblasts that expose mineral to osteoclasts and/or release a factor that activates these cells.
5. Activated osteoclasts dissolve the bone mineral and digest the organic matter of bone by the action of agents secreted in the segregated microcompartments underlying their ruffled borders. The mineral is solubilized by protons generated from CO, by carbonic anhydrase and secreted by an ATP-driven vacuolar H⁺-K⁺-ATPase located at the ruffled border. The organic matrix of the bone is removed by acid proteinases, particularly cysteine-proteinases that are secreted together with other lysosomal enzymes in the acid environment of the resorption zone.
6. Osteoclastic bone resorption is directly regulated by a polypeptide hormone, calcitonin (CT), and locally, by ionized calcium (Ca²⁺) generated as a result of osteoclastic bone resorption.
7. There is new evidence that osteoclast activity may also be influenced by the endothelial cells via generation of products including PG, NO and endothelin.     

Calcineurin/NFAT signaling in osteoblasts regulates bone mass

Development and repair of the vertebrate skeleton requires the precise coordination of bone-forming osteoblasts and bone-resorbing osteoclasts. In diseases such as osteoporosis, bone resorption dominates over bone formation, suggesting a failure to harmonize osteoclast and osteoblast function. Here, we show that mice expressing a constitutively nuclear NFATc1 variant (NFATc1(nuc)) in osteoblasts develop high bone mass. NFATc1(nuc) mice have massive osteoblast overgrowth, enhanced osteoblast proliferation, and coordinated changes in the expression of Wnt signaling components. In contrast, viable NFATc1-deficient mice have defects in skull bone formation in addition to impaired osteoclast development. NFATc1(nuc) mice have increased osteoclastogenesis despite normal levels of RANKL and OPG, indicating that an additional NFAT-regulated mechanism influences osteoclastogenesis in vivo. Calcineurin/NFATc signaling in osteoblasts controls the expression of chemoattractants that attract monocytic osteoclast precursors, thereby coupling bone formation and bone resorption. Our results indicate that NFATc1 regulates bone mass by functioning in both osteoblasts and osteoclasts.     

Imbalance of local bone metabolism in inflammatory arthritis and its reversal upon tumor necrosis factor blockade: direct analysis of bone turnover in murine arthritis

Chronic arthritis typically leads to loss of periarticular bone, which results from an imbalance between bone formation and bone resorption. Recent research has focused on the role of osteoclastogenesis and bone resorption in arthritis. Bone resorption cannot be observed isolated, however, since it is closely linked to bone formation and altered bone formation may also affect inflammatory bone loss. To simultaneously assess bone resorption and bone formation in inflammatory arthritis, we developed a histological technique that allows visualization of osteoblast function by in-situ hybridization for osteocalcin and osteoclast function by histochemistry for tartrate-resistant acid phosphatase. Paw sections from human tumor necrosis factor transgenic mice, which develop an erosive arthritis, were analyzed at three different skeletal sites: subchondral bone erosions, adjacent cortical bone channels, and endosteal regions distant from bone erosions. In subchondral bone erosions, osteoclasts were far more common than osteoblasts. In contrast, cortical bone channels underneath subchondral bone erosions showed an accumulation of osteoclasts but also of functional osteoblasts resembling a status of high bone turnover. In contrast, more distant skeletal sites showed only very low bone turnover with few scattered osteoclasts and osteoblasts. Within subchondral bone erosions, osteoclasts populated the subchondral as well as the inner wall, whereas osteoblasts were almost exclusively found along the cortical surface. Blockade of tumor necrosis factor reversed the negative balance of bone turnover, leading to a reduction of osteoclast numbers and enhanced osteoblast numbers, whereas the blockade of osteoclastogenesis by osteoprotegerin also abrogated the osteoblastic response. These data indicate that bone resorption dominates at skeletal sites close to synovial inflammatory tissue, whereas bone formation is induced at more distant sites attempting to counter-regulate bone resorption.     

Imbalance of local bone metabolism in inflammatory arthritis and its reversal upon tumor necrosis factor blockade: direct analysis of bone turnover in murine arthritis

Chronic arthritis typically leads to loss of periarticular bone, which results from an imbalance between bone formation and bone resorption. Recent research has focused on the role of osteoclastogenesis and bone resorption in arthritis. Bone resorption cannot be observed isolated, however, since it is closely linked to bone formation and altered bone formation may also affect inflammatory bone loss. To simultaneously assess bone resorption and bone formation in inflammatory arthritis, we developed a histological technique that allows visualization of osteoblast function by in-situ hybridization for osteocalcin and osteoclast function by histochemistry for tartrate-resistant acid phosphatase. Paw sections from human tumor necrosis factor transgenic mice, which develop an erosive arthritis, were analyzed at three different skeletal sites: subchondral bone erosions, adjacent cortical bone channels, and endosteal regions distant from bone erosions. In subchondral bone erosions, osteoclasts were far more common than osteoblasts. In contrast, cortical bone channels underneath subchondral bone erosions showed an accumulation of osteoclasts but also of functional osteoblasts resembling a status of high bone turnover. In contrast, more distant skeletal sites showed only very low bone turnover with few scattered osteoclasts and osteoblasts. Within subchondral bone erosions, osteoclasts populated the subchondral as well as the inner wall, whereas osteoblasts were almost exclusively found along the cortical surface. Blockade of tumor necrosis factor reversed the negative balance of bone turnover, leading to a reduction of osteoclast numbers and enhanced osteoblast numbers, whereas the blockade of osteoclastogenesis by osteoprotegerin also abrogated the osteoblastic response. These data indicate that bone resorption dominates at skeletal sites close to synovial inflammatory tissue, whereas bone formation is induced at more distant sites attempting to counter-regulate bone resorption.     

Signal transduction, receptors, mediators and genes: younger than ever - the 13th meeting of the Signal Transduction Society focused on aging and immunology

Background

The purpose of this study is to determine whether isolated suspension mouse peripheral mononucleated blood cells have the potential to differentiate into two distinct types of cells, i.e., osteoblasts and osteoclasts.

Results

Differentiation into osteoblast cells was concomitant with the activation of the Opn gene, increment of alkaline phosphatase (ALP) activity and the existence of bone nodules, whereas osteoclast cells activated the Catk gene, increment of tartrate resistant acid phosphatase (TRAP) activity and showed resorption activities via resorption pits. Morphology analyses showed the morphology of osteoblast and osteoclast cells after von Kossa and May-Grunwald-Giemsa staining respectively.

Conclusions

In conclusion, suspension mononucleated cells have the potentiality to differentiate into mature osteoblasts and osteoclasts, and hence can be categorized as multipotent stem cells.     

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.     

A new member of tumor necrosis factor ligand family, ODF/OPGL/TRANCE/RANKL, regulates osteoclast differentiation and function

Osteoclasts, the multinucleated giant cells that resorb bone, develop from monocyte-macrophage lineage cells. Osteoblasts or bone marrow stromal cells have been suggested to be involved in osteoclastic bone resorption. The recent discovery of new members of the tumor necrosis factor (TNF) receptor-ligand family has elucidated the precise mechanism by which osteoblasts/stromal cells regulate osteoclast differentiation and function. Osteoblasts/stromal cells express a new member of the TNF-ligand family "osteoclast differentiation factor(ODF)/osteoprotegerin ligand (OPGL)/TNF-related activation-induced cytokine (TRANCE)/receptor activator of NF-kB ligand (RANKL)" as a membrane associated factor. Osteoclast precursors which possess RANK, a TNF receptor family member, recognize ODF/OPGL/TRANCE/RANKL through cell-to-cell interaction with osteoblasts/stromal cells, and differentiate into osteoclasts in the presence of macrophage colony-stimulating factor. Mature osteoclasts also express RANK, and their bone-resorbingactivity is also induced by ODF/OPGL/TRANCE/RANKL which osteoblasts/stromal cells possess. Osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF)/TNF receptor-like molecule 1 (TR1) is a soluble decoy receptor for ODF/OPGL/TRANCE/RANKL. Activation of NF-kB and c-Jun N-terminal kinase through the RANK-mediated signaling system appears to be involved in differentiation and activation of osteoclasts.     

High level secretory expression of murine OCIL by CHO cells and action of OCIL on osteoclast differentiation

Receptor activator of nuclear factor-kB ligand (RANKL), a well-known membrane-bound molecule expressed on osteoblasts and bone marrow stromal cells, is believed to induce osteoclast differentiation and activation by binding to the receptor activator of nuclear factor-kB (RANK), which is expressed on the surface of osteoclast lineage cells. This induction is inhibited by osteoprotegerin (OPG) that is secreted by osteoblast lineage and acts as a decoy receptor of RANKL. Currently the essential role of the OPG/RANKL/RANK system in the process of osteoclast maturation, as well as activation, has been well established, and the majority of bone resorption regulators control osteoclast formation and activation through their effects on this system and especially on the relative expression levels of RANKL and OPG [1].     

Tumor necrosis factor-alpha induces differentiation of and bone resorption by osteoclasts

Osteoclast progenitors differentiate into mature osteoclasts in the presence of receptor activator of NF-kappaB (RANK) ligand on stromal or osteoblastic cells and monocyte macrophage colony-stimulating factor (M-CSF). The soluble RANK ligand induces the same differentiation in vitro without stromal cells. Tumor necrosis factor-alpha (TNF-alpha), a potent cytokine involved in the regulation of osteoclast activity, promotes bone resorption via a primary effect on osteoblasts; however, it remains unclear whether TNF-alpha can also directly induce the differentiation of osteoclast progenitors into mature osteoclasts. This study revealed that TNF-alpha directly induced the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs), which produced resorption pits on bone in vitro in the presence of M-CSF. The bone resorption activity of TNF-alpha-induced MNCs was lower than that of soluble RANK ligand-induced MNCs; however, interleukin-1beta stimulated this activity of TNF-alpha-induced MNCs without an increase in the number of MNCs. In this case, interleukin-1beta did not induce TRAP-positive MNC formation. The osteoclast progenitors expressed TNF receptors, p55 and p75; and the induction of TRAP-positive MNCs by TNF-alpha was inhibited completely by an anti-p55 antibody and partially by an anti-p75 antibody. Our findings presented here are the first to indicate that TNF-alpha is a crucial differentiation factor for osteoclasts. Our results suggest that TNF-alpha and M-CSF play an important role in local osteolysis in chronic inflammatory diseases.