Modulation of Osteoclast Differentiation and Function by the New Members of the Tumor Necrosis Factor Receptor and Ligand Families

I. Introduction II. Role of Osteoblasts/Stromal Cells in Osteoclast Differentiation and Function A. Origin of osteoclasts B. Stimulation of osteoclast differentiation by osteoblasts/stromal cells C. Stimulation of osteoclast function by osteoblasts/stromal cells III. New Members of the Tumor Necrosis Factor (TNF) Receptor and Ligand Families A. Osteoprotegerin (OPG) B. Osteoclast differentiation factor (ODF) and stromal osteoclast-forming activity (SOFA) IV. Regulatory Mechanism in Osteoclast Development and Function A. Regulatory mechanism of osteoclast differentiation by RANKL B. Regulatory mechanism of RANKL action on osteoclast function C. Signals induced by interleukin-1 (IL-1) and RANKL in osteoclasts V. Regulation of Human Osteoclast Development VI. Summary and Conclusion OSTEOCLASTS, which are present only in bone, are multinucleated giant cells with the capacity to resorb mineralized tissues. During the past decade, several new approaches have been developed to investigate osteoclast biology. A coculture system of mouse osteoblasts/stromal cells and hemopoietic cells for osteoclast formation has established the concept that osteoblasts/stromal cells are crucially involved in osteoclast development. Cell-to-cell contact between cells of the osteoblast lineage and hemopoietic cells is necessary for inducing differentiation of osteoclasts. It has been proposed that osteoblasts/stromal cells express osteoclast differentiation factor (ODF) or stromal osteoclast forming activity (SOFA) as a membrane-associated factor in response to several osteotropic factors such as 1α,25-dihydroxyvitamin D3[ 1α,25(OH)2D3], PTH, and interleukin 11 (IL-11). Osteoclast precursors of the monocyte-macrophage lineage recognize ODF/SOFA through cell-to-cell interaction with osteoblasts/stromal cells, and then differentiate into osteoclasts. Osteoblasts/stromal cells also play an essential role in the activation of osteoclast function. We emphasize that the term“ osteoblasts/stromal cells” is an operational one, used for convenience to describe those cells of the osteoblast lineage that have been shown convincingly in vitro to determine osteoclast formation. It is not certain in vivo which members of the lineage cells are responsible. In vitro data suggest that the osteoblast property is progressively lost with maturation of the osteoblast lineage cells, and in vivo, it is not at all likely that mature, synthesizing osteoblasts make any contribution to osteoclast formation. Nor are osteocytes likely to do so, but likely potential contributors are lining cells and early members of the osteoblast lineage that are situated close to the endosteal surface. Ultimately, the process of osteoclast formation is dependent on hemopoietic precursors being presented to the appropriate osteoblasts/stromal cells in an environment that provides appropriate stimulatory factors. Recently, three laboratories independently cloned cDNAs encoding the identical proteins, giving it the names osteoprotegerin (OPG), osteoclastogenesis inhibitory factor (OCIF), and tumor necrosis factor (TNF) receptor-like molecule 1 (TR1). This protein inhibits osteoclast development in vitro and in vivo. In an attempt to adopt a uniform nomenclature for this important biological activity, we propose that the name of choice be “osteoprotegerin.” OPG is a member of the TNF receptor family, but it does not have a transmembrane domain and possesses a signal sequence, suggesting that OPG functions as a secreted factor. Since OPG has the capacity to limit osteoclast formation, the ligand for this receptor was proposed to be the long-sought-after ODF/SOFA. Indeed, this hypothesis dictated the experiments carried out by the groups who subsequently identified a membrane-bound TNF-like ligand with the capacity to differentiate hematopoietic cells into functional osteoclasts. cDNA libraries from cell lines, which expressed specific binding sites for OPG, were screened by expression cloning approaches. As expected, the binding molecule of OPG was a membrane-associated protein of the TNF ligand family, which satisfied all the criteria of ODF/SOFA. In addition, ODF/SOFA was also able to maintain osteoclasts that had been induced by osteoblasts/stromal cells in an activated state. The discovery of this differentiation factor now opens a new era to investigate the molecular mechanism of osteoclast development and function. This review article describes the role of osteoblasts/stromal cells in osteoclast development and function at a molecular level, especially focusing on the central role of members of the TNF receptor and ligand superfamilies. Because discoveries in this area have originated from several directions and by different research groups, nomenclature has rapidly become confusing; thus, we propose an approach to overcome this. Development of osteoclasts proceeds within the local microenvironment of bone (1–4). This process can be replicated ex vivo using the coculture of mouse calvarial osteoblasts and spleen cells (5–9). Multinucleated cells formed in such cocultures satisfy the major criteria of osteoclasts such as tartrate-resistant acid phosphatase (TRAP, a marker enzyme of osteoclasts) activity, calcitonin receptors, p60c-src, vitronectin receptors (αvβ3), and the ability to form resorption pits on bone and dentine slices. Some mouse stromal cell lines such as MC3T3-G2/PA6 and ST2 resemble calvarial osteoblasts and support osteoclastogenesis in coculture with mouse spleen cells (10). Experiments on the osteopetrotic op/op mouse model have established that an osteoblast/stromal cell product, macrophage colony-stimulating factor (M-CSF, also called CSF-1), is crucial for osteoclast formation. The M-CSF gene of op/op mice cannot code functionally active M-CSF protein due to an extra thymidine insertion in the coding region of the M-CSF gene (11, 12). Administration of recombinant human M-CSF restored impaired bone resorption in op/op mice (13, 14). Calvarial osteoblasts obtained from op/op mice failed to support osteoclast development in cocultures with normal spleen cells, but the addition of M-CSF to cocultures induced osteoclast formation in response to 1α,25(OH)2D3 (15–17). These findings indicate that M-CSF produced by osteoblasts/stromal cells plays an essential role in osteoclast development. After identification of the hemopoietic origin of osteoclasts, much attention has been focused on the cell lineages of osteoclast progenitors. Using ST2 cells as a stromal supportive cell line, it was shown that, in addition to spleen cells and bone marrow cells, peripheral blood mononuclear cells and alveolar macrophages acted as a source of osteoclast precursors (18). Chambers et al. (19) have established cell lines that express macrophage from et al. have also established the cell from a These cell lines into osteoclasts were with stromal cells in the of that osteoclasts are from cells of the et al. have shown that osteoclasts formed from of and macrophage the in human marrow et al. have that mouse cells also form osteoclasts in the These indicate that osteoclast precursors are from cells in the monocyte-macrophage with as the the of the gene and gene in mice the origin of osteoclasts. of the to osteopetrotic in bone by a in osteoclast of normal bone marrow cells into mice the In addition, the of and macrophages was in of suggesting that the of the gene a lineage between osteoclast and macrophage differentiation In cocultures of osteoblasts and spleen cells, spleen cells failed to differentiate into osteoclasts. is a and and mice were to be osteopetrotic The development of osteoclasts and macrophages was in The osteopetrotic of mice was by of normal bone marrow cells into the The of macrophages and osteoclasts in mice that this factor the of These support the that osteoclasts are from cells of the the mechanism by which osteoclast and the is not at In the coculture cell-to-cell contact between osteoblasts/stromal cells and hemopoietic cells was to be for osteoclast development the osteoblasts/stromal cells have been identified as the cells for osteotropic and M-CSF to osteoclast development. activity a cell receptor that of a receptor and a but protein A which transmembrane and was to the through in response to recombinant induced osteoclast formation in the but osteoclasts were formed in response to in the of This that a by is involved in osteoclast development. such as and inhibitory which through also induced osteoclast formation in coculture experiments in mice human the expression of in osteoblasts was shown to be for of osteoclast osteoblasts obtained from human mice were with normal spleen cells, osteoclast formation was induced in response to human human This that that as a signal on osteoblasts/stromal cells but not on osteoclast to osteoclast formation. are present in mice of the of as of the major used to osteoclast formation is with the for this being The cells of in inducing osteoclasts are also osteoblasts/stromal cells but not osteoclast in the of the human cell were established to human protein receptor a cell lines, and which expressed functional recombinant osteoclast formation in response to in the coculture with mouse spleen cells, the cells not of the for to be expressed on the osteoblast was using cocultures established between osteoblasts and spleen cells from normal and mice It was shown that osteoclasts were formed in response to in cocultures of spleen cells obtained from mice and normal calvarial osteoblasts These indicate that the expression of in osteoblasts/stromal cells is for osteoclast formation in the The used for osteoclast is that by and have in 1α,25(OH)2D3 receptor mice by of the obtained from mice failed to support osteoclast development in cocultures with normal spleen cells in response to 1α,25(OH)2D3 but in response to In spleen cells from mice into osteoclasts in coculture with normal osteoblasts in response to These suggest that the by and are also into osteoblasts/stromal cells to osteoclast formation in the The normal osteoclast formation in mice is by the the induced by all factors are in osteoblasts/stromal cells to osteoclast formation this in we have proposed the hypothesis that osteoblasts/stromal cells express which is a membrane-bound factor to differentiation of osteoclast into osteoclasts through a mechanism cell-to-cell contact (5–9). Chambers et al. (19) also proposed that expressed by osteoblasts/stromal cells is involved in osteoclast and are the to that were to be and the concept of of osteoclast formation. A concept of osteoclast factors such as PTH, and osteoclast formation in cocultures of osteoblasts/stromal cells and hemopoietic cells for factors are osteoblasts/stromal different by and or stromal osteoclast forming activity (SOFA) as a membrane-associated factor in osteoblasts/stromal Osteoclast of the monocyte-macrophage lineage recognize ODF/SOFA in osteoblasts/stromal cells through cell-to-cell and then differentiate into osteoclasts. M-CSF produced by osteoblasts/stromal cells is a for and differentiation of osteoclast progenitors. to the of osteoclast function is that to to formed on are to by with or or We have developed a of mouse bone marrow cells and osteoblasts to functionally active osteoclasts The of osteoclasts in this was only with osteoblasts the osteoclast this osteoclast to be a source by which to a resorption system using dentine This established a to determine the ability of osteoclasts by the property of bone or In we were able to osteoclasts by the osteoclast on a osteoclasts for on dentine failed to form resorption of osteoclasts was restored calvarial osteoblasts were to the osteoclasts Some stromal cell lines such as and ST2 also activity of osteoclasts. cell-to-cell contact between osteoblasts/stromal cells and osteoclasts was osteoclasts failed to form resorption osteoblasts/stromal cells to play an essential role not only in inducing osteoclast formation from but also in to osteoclasts to are cell-to-cell In has that cells are formed from human and hemopoietic in the of osteoblasts/stromal et al. have also used to mononuclear or osteoclasts from cocultures of mouse bone marrow cells and osteoblasts These cells expressed of the of osteoclasts such as calcitonin receptors, and vitronectin only resorb bone cells and 1α,25(OH)2D3 were These support the hypothesis that cells of the osteoblast lineage osteoclast function. In et al. the discovery (OPG) that bone OPG of acid was a member of the TNF receptor family, all members of the family, a transmembrane domain and a secreted TNF expression of OPG in mice in an et al. independently the protein osteoclastogenesis inhibitory factor as a protein from the of human and that cDNA was identical to that of OPG OPG osteoclast formation induced by PTH, or in the In vivo of OPG in an in bone and bone with a of active osteoclast in normal and was also by OPG into et al. also identified a new member of the TNF receptor receptor-like molecule from a of an expressed data was to be identical to OPG and osteoclast formation in the coculture formation by osteoclasts, and bone resorption in of mouse and to a uniform nomenclature we propose the (OPG) be for the that and A of the and receptor of the new TNF involved in osteoclast formation. for the are We to propose that and OPG be as the names of the signal and receptor for the new TNF family, members of the TNF receptor OPG In addition, OPG had domain by a with a and with of TNF receptor and which of human OPG have been using The of OPG was to osteoclast formation in the the of the protein that was to of that of the A was in and the for not with the OPG can as a or as a of formation of a using present in of OPG was not necessary for biological activity of OPG the of the to not the inhibitory activity of In addition, the transmembrane domain of was between and and the protein was expressed in the human cell was induced in the cells the role of and of OPG is not the are active in A of functional of Human OPG is of acid in domain in is for formation of OPG is also of acid with identical to human The of OPG have been in mice produced by of the gene mice were and but by osteoclast formation and function. of and of bone with an in the of osteoclasts were in of The and of were that the osteoblast as as the osteoclast were in mice mice also developed of the and These indicate that OPG is a of bone resorption bone It also that OPG of is a ligand that binding to receptors, and et al. that OPG to and of also that the activity of OPG in the These indicate a potential mechanism OPG and which also in the of bone As OPG was a member of the TNF receptor family, a likely for ODF/SOFA be a membrane-bound ligand for this Since ODF/SOFA be expressed on the of ST2 cells 1α,25(OH)2D3 and this cell was for the ability of OPG to to ST2 cells with 1α,25(OH)2D3 and cloning of the ligand for OPG was using a cDNA of ST2 A cDNA with an encoding acid was The molecule was a transmembrane protein of the TNF ligand cells with the molecule expression were with and with mouse spleen cells in the of osteoclasts were formed on the This that the molecule cell-to-cell for A form of the protein with M-CSF induced osteoclast formation from spleen cells in the of osteoblasts/stromal cells, which was by OPG of calvarial osteoblasts with the of osteoclast formation, PTH, or expression of of this molecule it was that the molecule was which an essential signal to osteoclast for differentiation into osteoclasts. the molecule was called In to the stimulatory of 1α,25(OH)2D3 and on OPG were suggesting that the of OPG is also for osteoclastogenesis induced by osteotropic factors. A of the of and the of on osteoclast formation in mouse spleen cell is of acid The transmembrane between and The domain in are and in the TNF ligand the of which is to the of to the used in this spleen cells cells were in in the or of and After for the of multinucleated cells three or were as osteoclasts. are expressed as the of three of mouse with a form of also the of from the bone which was by OPG OPG, bone resorption in induced by not only but also by PTH, and These indicate that bone resorption induced by osteotropic factors is by et al. also in the molecular cloning of a ligand for OPG from an expression of the cell The OPG ligand was identical to expressed by human in membrane-associated and is that the form of (ODF) is present in the microenvironment of A recombinant form of osteoclast development in bone marrow in the of and it induced formation by M-CSF in an of bone marrow activity of osteoclasts from was also by was into mice a for was induced the of osteoclasts was identical to those of mice These indicate that not only osteoclast differentiation but also osteoclast function. Recently, et al. have in mice with of bone marrow within endosteal mice osteoclasts but have normal osteoclast that can differentiate into functionally active osteoclasts with normal osteoblasts/stromal In addition, mice and have a in These suggest that is an for osteoclast and it plays an important role in cell differentiation as The molecular cloning of that this molecule was identical to and receptor of factor which were independently identified by groups as a member of the TNF ligand was cloned a for in cell that and expressed of A recombinant form of induced activation of in The receptor was on cells by the receptor for to be dependent on TNF factor was impaired in from mice the of mouse and human cells in vitro with of expression The in the of cells induced by was by an in cell in a of and ligand et al. cloned a new member of the TNF receptor from a cDNA of human The mouse was also from the mouse cDNA The mouse cDNA a transmembrane protein of acid OPG, this protein of human expression of with in the and failed to members of the TNF ligand such as or In for the binding molecule of a ligand was cloned from a cDNA of cells and to be identical to A form of RANKL the ability of cells to cell in a and the of et al. that and with at the acid of in human cells and factor the necessary for the binding was the receptor was of activity but not This that interaction with is necessary for activation but not for the activation of the et al. that was also with the of the domain in addition to the in the receptor of and receptor activation in the cells These suggest that differentiation and activation of osteoclasts through by These findings to be to the that the was impaired in from mice are for the of The and receptor of the new TNF members are in and RANKL are the molecule important for development and function of cells and cells as as osteoclasts. to be the receptor for is a receptor for and to function as a These and receptors and have a of functions and on cells osteoclasts and In the of and the nomenclature of to be is only the and we that this name be it be that an identical molecule that function has been proposed as the receptor The ligand for has been as and and in RANKL as the nomenclature for this molecule we into and The of that biological are specific to such a name be to the bone it is that be by action on and cells, and functions in tissues. of indicate that the molecule biological by binding to OPG, but OPG also to OPG to function as a receptor as have The from suggesting that this molecule is expressed only activation of is for expression of this which be We have several for that RANKL be the this molecule is to the only ligand identified for the membrane-bound it does not a or it describes the of the is giving rapidly to of this the of we the names of OPG, and subsequently in this review article M-CSF and RANKL are the essential factors for inducing osteoclasts from mouse hemopoietic had colony-stimulating activity in a of bone marrow cells, and it not the formation. This that RANKL is not a factor but a differentiation factor of osteoclast progenitors. Using and OPG, the process of osteoclast differentiation was in In the coculture the can be into the in which of osteoclast and the in which differentiation into osteoclasts is was to the coculture for the cells on in the of In to the cocultures the of osteoclast but not differentiation into osteoclasts in response to the of M-CSF in osteoclast normal spleen cells were with osteoblasts from op/op mice M-CSF was the coculture osteoclasts were formed in response to the of M-CSF for the or for the failed to in osteoclast formation. These that M-CSF is for the and the differentiation of osteoclast development et al. that M-CSF plays important in and differentiation of osteoclast in mouse bone marrow The differentiation of osteoclast into functionally active osteoclasts and the for of the Recently, we osteoclast precursors obtained from cocultures osteoclast precursors expressed of the such as and and into osteoclasts cell in the of osteoclast precursors were with and into multinucleated cells within in the of These also that M-CSF and RANKL are involved in the differentiation of osteoclast precursors into osteoclasts osteoblasts/stromal cells were from the osteoclasts rapidly within by several and and M-CSF the of osteoclasts the of and function of osteoclasts by and were using from cocultures of mouse cells and bone marrow within and M-CSF and of through receptors The of on were by the of receptor but not by a M-CSF receptor The on but not resorption activity of on dentine was induced by in the of osteoblasts/stromal M-CSF failed to formation in the on dentine slices. As osteoclasts from the coculture failed to form resorption activity of osteoclasts was by but not by in the of osteoblasts/stromal it is that and M-CSF and of but only to osteoclasts that are active in resorption These suggest that play a role as a of osteoclast activation in bone of osteoclasts was also by of osteoclasts with OPG the of osteoclasts by but not that by or and the and of In addition, induced the activity of These indicate that RANKL and to osteoclast function in the of osteoblasts/stromal cells This was by the experiments using osteoclast in which osteoblasts were marrow cells were on in the of and M-CSF but in the of osteoblasts/stromal formed were by osteoclasts were on dentine rapidly forming resorption and all the of those osteoclasts, but only and induced the osteoblasts were to osteoclasts, resorption pits were factors such as PTH, and formation only in the of osteoblasts from op/op mice also induced activity of osteoclasts, which was by These support the hypothesis that osteoblasts/stromal cells osteoclast function through RANKL as a membrane-associated factor. RANKL can be with to and activation of osteoclasts. not support differentiation of osteoclast precursors into in the of osteoblasts/stromal cells were These also suggest that RANKL is involved in bone is involved in bone resorption such as and RANKL has been shown to resorption by osteoclasts the of activation of osteoclasts is by using a system in which osteoclast formation We of osteoclasts by such a to determine that the of RANKL in osteoclasts from of cell as as a on osteoclast and activation of osteoclasts cannot be in vivo in the expressed by osteoblasts/stromal cells, out the of osteoclasts differentiation and that activated in the of osteoclasts, and the activation at addition formed in the cocultures have receptors The of which a with and the in the with the activation of The that a of was from the into all of the of the multinucleated osteoclasts. of osteoclasts with or to and of the of osteoclasts by These indicate that the of osteoclasts through activation that osteoclasts formed in the cocultures expressed of osteoclasts with activated within which was by the of and also activated within in the osteoclasts. These suggest that the activation of and in osteoclasts by and in of osteoclast activation expression in the osteoclasts with suggesting that a is involved in the of osteoclasts. Signals induced by and RANKL in osteoclasts. formed in the cocultures express receptor and and RANKL the and activation of osteoclasts in the of osteoblasts/stromal and RANKL and through receptor and OPG and Recently, et al. and et al. independently mice in and of The mice developed of a in osteoclast The osteopetrotic was by bone marrow that the osteoclast were were but the of macrophages was in bone from the These suggest that and can be with in formation with in osteoclast RANKL has been to in the cells and we have also this in the osteoclast These suggest that the activation of in osteoclast also plays a crucial role in differentiation into osteoclasts. It is also that factors are by in osteoclast precursors and osteoclasts. As RANKL and M-CSF are essential factors for mouse osteoclast formation. findings indicate that the mechanism of human osteoclast formation is to that of mouse osteoclast formation. et al. that cells and ST2 cells human osteoclast formation in coculture with human peripheral blood mononuclear cells in the of 1α,25(OH)2D3 and In addition of human M-CSF to the coculture was essential to human osteoclasts and ST2 cells and mouse which do not to human M-CSF receptors This also that and mouse RANKL can on human cells as We also that the human cell line, which expressed functionally active receptors, human osteoclast formation in response to and in coculture with human peripheral blood mononuclear cells M-CSF mouse and human osteoclast formation in coculture with These are with the of and who a role of M-CSF in human osteoclast formation as of human with mouse and human M-CSF with induced human osteoclasts OPG osteoclast formation from human that was by cells or by human induced expression of RANKL by cells, and this was not by These suggest that cells in human an inhibitory human of which is by osteoclasts were from human that had been on a and M-CSF were the for colony-stimulating factor has been shown to be an important factor for osteoclast formation in human bone marrow as in the of mouse osteoclast formation, human osteoclast formation induced by and M-CSF and This that of osteoclast but inhibits differentiation into osteoclasts. These also indicate that of human osteoclast formation are the as those of mouse osteoclast formation. Regulation of human osteoclast formation and function can be from the findings obtained from the mouse with human human osteoclast formation. Human were in cells in the or of human mouse human OPG, human and After for multinucleated cells three or were as osteoclasts. Human were in cells with or human M-CSF and mouse in the of After for cells were for Human were in cells in which a dentine had been were with human M-CSF and mouse in the of After for resorption pits formed on the were with Osteoblasts/stromal cells are involved in osteoclast differentiation and function through cell-to-cell contact have been to the mechanism of the by osteoblasts/stromal it has an OPG and binding molecule were The discovery of the new members of the TNF members has the that osteoclast differentiation and function are by osteoblasts/stromal which has also been called or is a member of the TNF ligand of RANKL in osteoblasts/stromal cells is by osteotropic factors such as PTH, and Osteoclast precursors express a TNF receptor recognize RANKL through cell-to-cell interaction with osteoblasts/stromal cells, and differentiate into in the of RANKL is also involved in the and of and activation of osteoclasts. OPG, which has also been called or is a receptor for RANKL and as a receptor in the system A of osteoclast differentiation and function by osteoblasts/stromal In osteoblasts/stromal cells are involved in all of the of osteoclast such as and activation of osteoclasts Osteoblasts/stromal cells can now be with RANKL and M-CSF in with the of osteoclasts. and OPG are three that osteoclast and function. on the molecular mechanism of the of bone This of new to several bone by osteoclast and functions such as bone and bone We and of and and of for of the and