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Department of Oral Cell Biology, Umeå University, 901 87 Umeå, Sweden; ulf.lerner{at}odont.umu.se
Abstract (1) Introduction (2) Osteoclast Differentiation (3) Importance of Osteoblast/Stromal Cells in Osteoclast Differentiation (4) Receptor Activator of Nuclear Factor B Ligand
(5) Receptor Activator of Nuclear Factor (6) Osteoprotegerin (7) Downstream Intracellular Signaling Molecules (8) RANKL-RANK-OPG in Dental Tissues (9) RANKL/RANK/OPG System in Pathological Conditions (10) Summary Acknowledgments REFERENCES
| Abstract |
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Key words. RANKL, OPG, osteoclasts, periodontal disease, osteopetrosis, osteoporosis
| (1) Introduction |
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The physiological remodeling of bone takes place in so-called ‘bone multi-cellular units’ (BMUs) and is initiated by recruitment, formation, and activation of bone-resorbing osteoclasts. These cells resorb a given volume of bone, and, subsequently, the Howship’s resorption lacunae are filled with new bone formed by recruited and activated osteoblasts. Such BMUs are present both at the surfaces of trabecular and cortical bone and in the Haversian canals of cortical bone but are more frequent in trabecular bone. This is the reason a metabolic bone disease such as osteoporosis primarily affects bones with a large proportion of trabecular bone. It has been estimated that, under normal conditions, 1 or 2 x 106 BMUs are present in the adult skeleton.
Since remodeling of bone in pathological conditions mostly results in increased bone resorption, much of the interest in bone cell biology has been focused on osteoclasts. Many different systemic hormones and local growth factors/cytokines have been demonstrated to stimulate or inhibit osteoclast formation and/or function. Also, recently, to this list of osteotropic factors have been added the signaling molecules present in the peripheral nervous system and mechanisms controlled by the central nervous system (Baldock et al., 2002; Lerner and Lundberg, 2002. Takeda et al., 2002).
Already during the late 1970s, it was shown that osteoclasts are derived from haematopoietic tissues and are in fact leukocytes. We now know that osteoclasts are formed by fusion of mononuclear progenitors of the monocyte-macrophage lineage. During the last decade, it has become evident that osteoblasts in the periosteum, and osteoblast-like stromal cells in hematopoietic tissues, control osteoclast formation/activation via cell-to-cell contacts with the progenitor cells. During past years, the molecules responsible for the interaction between these cells have been found to be members of the tumor necrosis factor (TNF) ligand and receptor superfamilies. Thus, the expression of receptor activator of nuclear factor-B ligand (RANKL) on the surfaces of stromal cells/osteoblasts and the activation of its cognate receptor RANK on the surfaces of hematopoietic cells are required for osteoclast formation and activation (Fig. 1
). This interaction can be blocked by the soluble ‘decoy’ receptor osteoprotegerin (OPG) secreted by stromal cells/osteoblasts. Mainly due to expected, and sometimes unexpected, phenotypic findings in mice with specific targeted gene deletions, we have also acquired information on the critical importance for the differentiation and function of osteoclasts of other molecules expressed in stromal cells/osteoblasts and osteoclast progenitor cells. It is the aim of the present review to summarize, briefly, the knowledge about the molecules involved in osteoclast differentiation and activation and to focus, in more detail, on the TNF ligand and receptor-like molecules.
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| (2) Osteoclast Differentiation |
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). Mice deficient in the transcription factor PU.1 lack both macrophages and osteoclasts, because of the absence of common early precursor cells for macrophages and osteoclasts (Tondravi et al., 1997). Consequently, the osteopetrotic phenotype of PU.1–/– mice can be rescued by bone marrow transplantation.
Activation of the c-fms receptor by its cognate ligand macrophage colony-stimulating factor (M-CSF) is necessary for the proliferation and survival of macrophages/osteoclast progenitor cells, and loss of function mutation in the M-CSF gene in op/op mice is the reason for the lack of osteoclasts in these mice and their osteopetrotic phenotype (Yoshida et al., 1990). Since M-CSF is expressed by stromal cells/osteoblasts, op/op mice are not cured by bone marrow transplantation. The signaling pathway of M-CSF includes mitogen-activated protein kinases (MAPK)-induced phosphorylation of Mitf and TFE3, two closely related helix-loop-helix transcription factors of which Mitf has been linked to osteopetrosis in mi/mi mice (Weilbaecher et al., 2001). Interestingly, the osteopetrotic phenotype of the op/op mice is lost over time due to enhanced expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), indicating a redundancy of the M-CSF and GM-CSF genes.
The transcription factor AP-1 is a heterodimeric protein consisting of Fos proteins (c-Fos, FosB, Fra-1, and Fra-2) and Jun proteins (c-Jun, JunB, and JunD). Unexpectedly, mice deleted of the c-Fos gene exhibit an osteopetrotic phenotype due to lack of osteoclast progenitor cells (Wang et al., 1992; Grigoriadis et al., 1994). The osteopetrotic phenotype of c-Fos-deficient mice can be cured by bone marrow transplantation. The arrest of osteoclast formation in the c-Fos–/– mice is associated with increased numbers of macrophages, indicating that c-Fos acts downstream of PU.1. The lack of osteoclastogenesis in spleen cell cultures from c-Fos–/– mice can be rescued by in vitro transfection by all forms of Fos proteins, but not by Jun transfection, with Fra-1 having the highest activity (Matsuo et al., 2000). In line with these observations, transgenic mice expressing Fra-1 prevent the osteopetrotic phenotype of c-Fos-deficient mice. RANKL induces c-Fos-dependent transcription of Fra-1, indicating that Fra-1 is acting distal to c-Fos, which is also shown by the fact that Fra-1 expression is blunted in c-Fos–/– mice. Interestingly, RANKL-induced expression of interferon-ß (IFN-ß) in stromal cells/osteoblasts acts in a negative feedback manner and inhibits osteoclast formation by decreasing RANKL-stimulated c-Fos expression (Takayanagi et al., 2002a). The IFN-ß-induced inhibition of osteoclastogenesis can be rescued by suppressors of cytokine signaling (SOCS)-1 and-3, which also are up-regulated by RANKL (Hayashi et al., 2002). Mice which lack responsiveness to IFN-ß develop osteoporosis (Takayanagi et al., 2002a).
As described in detail below, deletions of the RANKL or RANK genes result in the absence of osteoclasts due to arrested differentiation of M-CSF expanded osteoclast progenitor cells. Deletion of the OPG gene, resulting in loss of the RANKL-inhibitory OPG, gives rise to mice with early-onset osteoporosis. The differentiation of osteoclast progenitor cells is also blocked by deletion of the gene for TNF receptor-associated factor 6 (TRAF6) (Naito et al., 1999), and by double knockout of the NF-B proteins p50 and p52 (Franzoso et al., 1997; Iotsova et al., 1997), which is not unexpected, since TRAF6 and NF-B are part of the RANK signaling pathway. The osteopetrotic phenotype of p50/p52–/– and TRAF6–/– mice can be overcome by bone marrow transplantation, indicating the importance of these molecules in the osteoclast progenitor cells. Recently, Battaglino et al. (2002), in a cDNA microarray screening project, found the proto-oncogene c-Myc to be a downstream target in RANK signaling in RAW 264.7 cells, and that expression of a dominant-negative Myc in these cells blocked RANKL-induced osteoclast formation.
The differentiation of mononuclear osteoclast progenitor cells to mature osteoclasts involves fusion to multinuclear cells and their polarization of the cell membrane adjacent to bone, required for development of the sealing zone and the ruffled border (Fig. 2). Attachment of osteoclasts to bone extracellular matrix in the sealing zone is associated with the presence of F-actin rings and has been suggested to be mediated by the integrin 
vß3, preferentially expressed in the sealing zone area, and the RGD sequence in osteopontin and bone sialoprotein. The importance of the vß3 expression is shown by the observations that RGD-containing peptides can block bone resorption in vitro, and that, although ß 3–/– mice can form multinucleated osteoclasts, the osteoclasts are less polarized, as assessed by a lack of actin rings and disorganization of the ruffled border (McHugh et al., 2000). The functional importance of these abnormalities is demonstrated by the facts that blood calcium is decreased, bone mass increased, and osteoclasts in ß3–/– mice are less effective in resorbing bone on dentin slices in vitro. Interestingly, it has been suggested that ligand binding to vß3 may not only be part of the attachment of osteoclasts to extracellular matrix, but also may be involved in intracellular signaling. Thus, vß3 activation is associated with changes of intracellular calcium and activation of c-src- and c-src-dependent phosphorylation of proline-rich tyrosine kinase-2 (Pyk-2). The latter is a kinase related to the focal adhesion kinase (FAK), which is suggested to be involved in formation of the sealing zone, as well as to the activation of phosphatidylinositol 3-hydroxyl kinase (PI3-kinase) (Hruska et al., 1995; Chellaiah et al., 1998; Duong et al., 1998; Zhang et al., 2002; Wang et al., 2003). The view that vß3 is not only of importance for anchoring osteoclasts to the extracellular matrix is supported by the observation that transfection of the full-length ß3-integrin gene rescues the impaired osteoclast function in ß3–/– mice, whereas transfection with a truncated form of ß3, lacking the cytoplasmic domain, does not have this activity (Feng et al., 2001). Interestingly, transfection with a construct containing the S752P substitution in the ß3 cytoplasmatic domain, characteristic of the bleeding disorder Glanzmann’s thrombasthenia, fails to restore the function of ß3–/– osteoclasts. The crucial role of ruffled border formation in osteoclast activity is illustrated by the finding that c-src-deficient mice have normal numbers of multinucleated osteoclasts, but the cells lack the ruffled border, and, as a consequence, the osteoclasts fail to resorb bone, and the c-src–/– mice exhibit an osteopetrotic phenotype (Soriano et al., 1991; Boyce et al., 1992). These mice can be cured by bone marrow transplantation.
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vß3 and osteopontin is important for bone resorption has also been examined in osteopontin-deficient mice. These mice exhibit no skeletal phenotype under normal conditions. In favor of the hypothesis, however, it has been found that osteopontin–/– mice are resistant to ovariectomy-induced bone loss (Yoshitake et al., 1999). Furthermore, bone resorption and osteoclast formation induced by PTH in cultured mouse calvariae are absent in calvariae from osteopontin knockout mice (Ihara et al., 2001). Thus, it is evident that osteopontin has a role in not only osteoclast polarization, but also in osteoclast recruitment. The molecular mechanism involved in this latter process is unknown. The bone-resorbing activity of a fully differentiated multinucleated osteoclast that is attached to bone and has developed a ruffled border involves mechanisms for dissolution of hydroxyapatite crystals and enzymatic degradation of bone matrix proteins. The process is initiated by secretion of protons mediated by a vacuolar type of H+-ATPase in the ruffled border. By this proton pump, a pH of ~ 4.5 is created in the microenvironment present in the Howship’s resorption lacunae, and bone mineral will be dissolved. The osteoclastic proton pump seems to contain a specific subunit, termed OC-116kD, which has been cloned. Targeted disruption of its gene (Atp6i) results in mice which can form osteoclasts, but the cells lose their capacity to generate extracellular acidification (Li et al., 1999). These mice exhibit a severe form of osteopetrosis. Atp6i–/– mice have normal intracellular lysosomal proton pump activity in osteoclasts and no disturbances in the liver lysosomes, or proton pump transport in kidney microsomes. Deletion in this gene has been found in the osteosclerotic mutant oc/oc mice (Scimeca et al., 2000). Several mutations in the human Atp6i gene, the vast majority of which result in loss of function, have recently been found in patients with autosomal-recessive osteopetrosis (Sobacchi et al., 2001; Michigami et al., 2002; Scimeca et al., 2003; Taranta et al., 2003).
To provide electroneutrality of acid secretion, chloride channels are present together with the H+-ATPase in osteoclastic ruffled-border membranes. Recently, the critical importance of the function of osteoclasts of one of the 9 known mammalian chloride channel genes was shown. Deletion of the gene for the ubiquitously expressed chloride channel ClC-7 resulted in mice with severe osteopetrosis and retinal degeneration (Kornak et al., 2001). Osteoclasts from ClC-7–/– mice differentiate normally and attach to bone surfaces, but are unable to create resorption lacunae because of a failure to secrete acid, although the H+-ATPase is expressed normally. Mutations in the human ClC-7 gene cause osteopetrosis in patients (Cleiren et al., 2001; Kornak et al., 2001; Campos-Xavier et al., 2003).
The demineralized bone in the resorption lacunae is degraded by proteolytic enzymes. The knowledge of which enzymes are involved in degradation of the different proteins in bone extracellular matrix is very limited. It is well-recognized, however, that cysteine proteinases play a crucial role, and, in general, cysteine proteinase inhibitors are potent inhibitors of bone resorption in vitro and in vivo. Cathepsin K is a cysteine proteinase with collagenolytic activity abundantly expressed in osteoclasts. Deletion of the cathepsin K gene results in mice with an osteopetrotic phenotype (Saftig et al., 1998; Gowen et al., 1999). In the resorption area beneath the osteoclasts, demineralized, but undegraded, bone matrix can be seen, indicating that the defect is primarily due to a lack of extracellular collagenolytic activity. This feature is also characteristic of resorption lacunae in the human osteopetrotic disease pycnodysostosis, which is linked to several mutations in the cathepsin K gene (Gelb et al., 1996). The possible role of matrix metalloproteinases (MMP) in osteoclastic resorption is less clear. An interesting hypothesis put forward by Delaisse and co-workers suggests that MMPs are required for the invasion of osteoclasts through the extracellular matrix, rather than for their activity in the Howship’s lacunae (Ensig et al., 2000). It has also been suggested that the role of MMPs in bone resorption is to be involved in ‘cleaning’, performed by the lining cells, of collagen fibrils left in the resorption lacunae by osteoclasts (Everts et al., 2002).
Recently, we have found that cysteine proteinases are involved not only in the bone-resorbing activity of osteoclasts, but also in osteoclast differentiation. Thus, cystatin C and a peptidyl derivative synthesized to mimic part of the proteinase-binding site of cystatin C (Z-RLVG-CHN2) inhibit osteoclast formation in mouse bone marrow cultures stimulated by PTH, D3, or IL-6 and in spleen cell cultures stimulated by M-CSF and RANKL. The effect is exerted at a late stage of the osteoclast progenitor cell differentiation. The enzyme(s) and mechanisms involved is/are presently unknown (Brage et al., 2004).
| (3) Importance of Osteoblast/Stromal Cells in Osteoclast Differentiation |
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). Gene deletion experiments have very elegantly demonstrated the indispensable role of osteoblasts, not only for osteoclast formation stimulated by PTH and D3, but also for the osteoclastogenic effects of interleukin-6 (IL-6) and prostaglandin E2 (PGE2). Thus, when osteoblasts isolated from PTH receptor (PTHR1) knockout mice, vitamin D3 receptor (VDR) knockout mice, or mice lacking the EP4 receptor for PGE2 were co-cultured with spleen cells from wild-type mice, no osteoclasts were formed in response to the ligand for these receptors (Liu et al., 1998; Takeda et al., 1999; Sakuma et al., 2000). In contrast, when spleen cells from PTHR1–/– mice, VDR–/– mice, or EP4–/– mice were co-cultured with osteoblasts from wild-type mice, osteoclasts were formed in response to PTH, D3, or PGE2. These observations demonstrate that it is the expression of the receptors for PTH, D3, and PGE2 in osteoblasts that is critical for their effects on osteoclastogenesis. However, PGE2 augments osteoclast formation in spleen cell cultures stimulated by RANKL, indicating that PGE2 may stimulate osteoclastogenesis via receptors expressed on both osteoblasts/stromal cells and osteoclast progenitors (Wani et al., 1999; X Li et al., 2000). The importance of prostaglandin EP2 and EP4 receptors—not only for the osteoclastogenic effect of PGE2, but also for those of PTH and D3—has been demonstrated (X Li et al., 2000; Tomita et al., 2002). IL-6 has been implicated as a cytokine involved in the stimulation of bone resorption in inflammatory conditions (e.g., rheumatoid arthritis, periodontitis), multiple myeloma, post-menopausal osteoporosis, and primary hyperparathyroidism. However, IL-6 stimulates neither osteoclast formation in mouse bone marrow cultures nor bone resorption in mouse calvariae (Tamura et al., 1993; Palmqvist et al., 2002). In contrast, other members of the IL-6 family of cytokines—such as IL-11, leukemia inhibitory factor (LIF), and oncostatin M (OSM)—stimulate osteoclastogenesis and bone resorption (Tamura et al., 1993; Palmqvist et al., 2002), demonstrating that it is not low expression of the signal-transducing protein gp130 that is the reason for the lack of effect by IL-6, but rather low expression of IL-6 receptors, shown to be true in mouse calvarial osteoblasts at the mRNA level (Palmqvist et al., 2002). However, when IL-6 is added to the soluble IL-6 receptor, both osteoclastogenesis and bone resorption are induced, and these effects can be inhibited by an antiserum neutralizing gp130. When spleen cells from wild-type mice are co-cultured with osteoblasts from transgenic mice constitutively expressing the human IL-6R, osteoclasts are formed in response to IL-6, even in the absence of soluble IL-6R (Udagawa et al., 1995). Since IL-6 does not stimulate osteoclastogenesis in spleen cell cultures from IL-6R transgenic mice, these findings also underscore the critical role of osteoblasts in osteoclast differentiation.
Although analysis of these data clearly indicates the importance of stromal cells/osteoblasts for osteoclastogenesis, it does not indicate by what molecular mechanisms osteoblasts control the development of osteoclasts. The observations in the osteopetrotic op/op mice of the role of M-CSF were the first to demonstrate the importance of an osteoblastic molecule for the formation of osteoclasts. The op/op mice exhibit an osteopetrotic phenotype due to lack of multinucleated osteoclasts. Co-cultures of spleen cells from op/op mice and wild-type osteoblasts are sensitive to the osteoclastogenic effect by D3, whereas co-cultures of spleen cells from wild-type mice and ob/ob osteoblasts are insensitive, indicating that the defect in the ob/ob mice is present in osteoblasts (Takahashi et al., 1991). The insensitivity of the latter co-cultures, as well as the in vivo phenotype, can be prevented by the addition of M-CSF (Felix et al., 1990; Komada et al., 1991; Takahashi et al., 1991). The demonstration in op/op mice of an extra thymidine insertion at base pair 262 in the coding region of the M-CSF gene, resulting in a TGA stop codon 21 base pairs downstream (Yoshida et al., 1990), clearly shows that the phenotype of op/op mice is due to functionally inactive M-CSF. Later it was shown that M-CSF is important for survival, proliferation, and differentiation of early osteoclast progenitor cells (Tanaka et al., 1993; Felix et al., 1994). However, M-CSF is a secreted molecule that interacts with its receptor c-fms expressed on osteoclast progenitor cells, and the critical role of this cytokine does not explain the requirement of cell-to-cell contact between stromal cells/osteoblasts and the osteoclast precursor cells. It was not until the very recent observations of the critical role of RANKL, expressed on the cell surfaces of stromal cells/osteoblasts, and its interaction with the receptor RANK, expressed on osteoclast progenitor cells, that the molecular mechanism involved in cell-to-cell contact was clarified. The activation of RANK by RANKL is inhibited by OPG, a soluble receptor with sequence homology to RANK, which is secreted by stromal cells/osteoblasts. The activation of RANK in osteoclast progenitor cells results in the commitment of the pool of M-CSF expanded precursors to differentiate into mature osteoclasts (see further below).
RANKL-RANK signaling is crucial not only for osteoclast progenitor cell differentiation but also for the activity of the mature, multinucleated osteoclasts (Fig. 2; Fuller et al., 1998).
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(4) Receptor Activator of Nuclear Factor B Ligand
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The cloning of RANKL revealed it to be a member of the TNF ligand superfamily, a cytokine family that also includes TNF-, TNF-ß, CD40 ligand, Fas ligand, CD30 ligand, TWEAK, and TRAIL (Locksley et al., 2001). Like other members of the TNF-like family of cytokines, RANKL is a type II membrane-embedded protein, with a large extracellular, receptor-binding domain, a membrane-anchoring domain, and a connecting stalk. The RANKL gene is present on human chromosome 13q14 and on mouse chromosome 14. The mouse and human RANKL consists of 316 and 317 amino acid residues, respectively. Sequence alignments of the amino acid residues among proteins in the TNF ligand family show quite a low level of amino acid conservation between the members. However, information from x-ray crystal structures has demonstrated several common features of the three-dimensional structure. One feature of the ligands in the TNF-like family is that they form self-assembling, non-covalent homotrimers with ß-pleated sheets assuming a ‘jellyroll’ orientation. The homology within the TNF ligand family is confined to domains involved in monomer folding and trimer assembly. The receptor-binding domains show only limited sequence homology between members of the TNF-like ligands, which is the basis for receptor selectivity. The shape of the ligand is that of an inverted bell, which at the base interacts with the receptors in 3:3 symmetric complex. Recently, the ectodomain of murine RANKL was crystallized, and it was shown that RANKL also self-associates as a homotrimer (Lam et al., 2001; Ito et al., 2002). The trimeric protein contains four unique surface loops that create the specificity in its interaction with the receptor RANK, elegantly demonstrated by site-directed mutagenesis of selected residues in these loops and functional osteoclastogenesis analysis with such mutated variants of recombinant RANKL in murine bone marrow cultures. Such structure-based comparative and functional studies will, in the future, be the basis for the rationale for and design of compounds affecting RANKL-induced osteoclastogenesis.
Like several other TNF-like type II proteins, RANKL trimers exist either as membrane-anchored proteins or in a soluble cleaved form, both being functionally active. Thus, the metalloprotease-disintegrin TNF- convertase (TACE), or a related protease, can release the ectodomain of RANKL (Lum et al., 1999), and the metalloprotease inhibitor KB-R8301 inhibits the release of soluble RANKL (Nakashima et al., 2000).
RANKL is most abundantly expressed in trabecular bone, bone marrow, growth plate, periosteum, spleen, thymus, lymph nodes, and intestinal lymphoid patches. At the cellular level, high expression levels of RANKL can be found in several different stromal cells/osteoblasts and lymphoid cell lines. RANKL expression can also be found in hypertrophic chondrocytes.
The expression of RANKL by stromal cells and osteoblasts is regulated by a variety of hormones and cytokines that stimulate osteoclast formation and bone resorption. RANKL can be induced by PTH, PGE2, and forskolin, all acting via the cyclic AMP/protein kinase A (PKA) pathway, and by D3, acting via the VDR-mediated pathway (Lerner, 2000; Teitelbaum, 2000; Hofbauer and Heufelder, 2001; Takahashi et al., 2002). Cytokines in the IL-6 family of cytokines, including IL-6 (in the presence of soluble IL-6 receptor), IL-11, OSM, and LIF (Horwood et al., 1998; Ahlen et al., 2002; Palmqvist et al., 2002), as well as IL-7 (Toraldo et al., 2003) and IL-17 (Nakashima et al., 2000), also increase RANKL expression in osteoblasts and stromal cells. The stimulatory effect of IL-11 is potentiated by heparin (Walton et al., 2002). In addition, RANKL expression in osteoblasts can be induced by histamin (Deyama et al., 2002), IGF-I (Rubin et al., 2002a), or activation of purinergic P2Y receptors by ATP (Buckley et al., 2002), and decreased by melatonin (Koyama et al., 2002) or mechanical stress (Rubin et al., 2002b). The bone-resorptive effects of PTH, D3, and the IL-6 family of cytokines can be blocked by the inhibitory decoy receptor OPG (Palmqvist et al., 2002), thereby demonstrating the crucial role of increased RANKL expression for their bone-resorptive effects. In addition, IL-1ß and TNF- increase RANKL mRNA in human osteoblastic cells (Hofbauer et al., 1999a). Conditional expression of dominant-negative forms of different transcription factors has shown that the stimulatory effect of PTH on RANKL expression is dependent on cyclic AMP response element-binding protein (CREB), but not on c-Fos or Cbfa1 (Fu et al., 2002). Dominant-negative expression of STAT-3 or gp130 in the stromal cell line UAMS-32 suppresses the osteoclast-supporting activity of these cells stimulated by IL-6 (plus sIL-6R), OSM, or IL-11, but not stimulation caused by PTH or D3 (O’Brien et al., 1999), suggesting the importance of the gp130/STAT-3 signaling pathway in the regulation of RANKL gene expression. Ionomycin, thapsigargin, and phorbol 12-myristate 13-acetate (PMA) have also been shown to stimulate RANKL mRNA, demonstrating that the calcium/PKC pathway is also linked to RANKL expression (Takami et al., 2000). Not only the VDR, but also other steroid hormone receptors can induce RANKL, and such an effect has been demonstrated for the glucocorticoid receptor (Hofbauer et al., 1999c; Lerner et al., unpublished), the retinoid receptor (Lerner et al., unpublished), and the thyroid receptor (Miura et al., 2002). We have demonstrated that the neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) inhibit osteoclastogenesis induced by PTH and D3, and that this effect is associated with decreased RANKL mRNA expression (Mukohyama et al., 2000).
The promoter region of the RANKL gene contains a response element for core-binding factor a1 (Cbfa-1; also known as AML-3, Pebp2aA, or Runx2), which is a transcription factor crucial for osteoblast differentiation and expression of bone matrix proteins (Ducy et al., 1997). Consequently, cbfa-1–/– mice have reduced mRNA expression of RANKL (Gao et al., 1998). In the periosteum of these mice, a few TRAP-positive osteoclasts can be seen, but they are smaller and have a reduced number of nuclei (Komori et al., 1997). In line with these observations, osteoblasts from cbfa-1–/– mice are less effective in supporting osteoclast formation when co-cultured with spleen cells from wild-type mice (Gao et al., 1998). Although cbfa-1 does not stimulate the 0.7-kb 5'-flanking region of the RANKL gene containing two putative binding sites for cbfa-1 (O’Brien et al., 2002), it was recently shown that adenoviral introduction of cbfa-1 into CA.120-4 cells induced RANKL expression (Enomoto et al., 2003). However, forced expression of RANKL in cbfa-1–/– mice did not completely restore osteoclast formation, suggesting that additional factors are missing in cbfa-1 knockout mice. The role of cbfa-1 in osteoclastogenesis was also demonstrated by the observation that transgenic mice overexpressing cbfa-1 under the control of the collagen (1) type I collagen promoter have a high bone turnover rate due to increased bone resorption and bone formation (Geoffroy et al., 2002). This phenotype is associated with increased formation of osteoclasts in bone marrow cultures treated with D3, as well as an increased number of osteoclasts in D3-stimulated co-cultures of transgenic osteoblasts with spleen cells from either wild-type or transgenic mice. The detailed knowledge of RANKL gene induction, however, has to await further analysis of the RANKL gene promoter region.
The crucial role of RANKL in osteoclastogenesis has been demonstrated in mice deficient in RANKL due to targeted deletion of the RANKL gene (Kong et al., 1999a). These mice exhibit a severe form of osteopetrosis due to complete absence of osteoclasts. Similar to many other forms of osteopetrosis, RANKL–/– mice have impaired tooth eruption. The lack of RANKL also results in disturbances in the growth plate, and the bones are shorter than normal. The osteopetrotic phenotype cannot be restored by bone marrow transplantation, which indicates that the lack of osteoclasts is not due to defective hematopoietic cells, a view which is in line with the fact that RANKL is expressed in stromal cells/osteoblasts. Although no TRAP-positive osteoclast progenitor cells can be seen in RANKL–/– mice, these mice have normal osteoclast progenitor cells, as demonstrated by the observation that osteoclast progenitor cells from RANKL–/– can differentiate to mature osteoclasts when co-cultured with osteoblasts from wild-type mice. In contrast, mature osteoclasts are not formed when osteoblasts from RANKL–/– are co-cultured with osteoclast precursors from wild-type mice (Kong et al., 1999a). Interestingly, in view of the very close relationship among osteoclasts, macrophages, and dendritic cells, the differentiation and function of macrophages and dendritic cells seem to be normal in RANKL-deficient mice. Similar to many osteopetrotic animals, RANKL–/– mice also have splenomegaly due to extramedullary hematopoiesis.
Mice overexpressing soluble RANKL under the control of the liver-specific human serum amyloid P component promoter exhibit increased numbers of osteoclasts and decreased bone mineral density (Mizuno et al., 2002). When soluble RANKL was expressed ubiquitously under the control of the ß-actin promoter, the mice died at the late fetal stage. Administration of RANKL for 7 days to rats also results in decreased bone mineral density, an effect observed in both cortical and trabecular bone (McHugh et al., 2003).
Besides the osteopetrotic phenotype, RANKL–/– mice exhibit impaired early T- and B-lymphocyte differentiation and a complete lack of lymph nodes (Kong et al., 1999a), which is likely to be related to RANKL/TRANCE expression in lymph nodes, spleen, thymus, and intestinal lymphoid patches (Wong et al., 1997; Lacey et al., 1998). However, spleen and Peyer’s patches seem to be normal. RANKL can be induced in mammary epithelial cells by injection of progesterone, prolactin, and parathyroid-hormone-related peptide (PTHrP), but not by estrogen (Fata et al., 2000). The regulation of RANKL by the pregnancy hormones indicates a role for this cytokine in mammary gland development required for lactation. In line with this hypothesis, Fata et al. (2000) also reported that RANKL–/– female mice fail to form lobulo-alveolar mammary structures during pregnancy, leading to the death of newborn pups.
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(5) Receptor Activator of Nuclear Factor B
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RANK can be demonstrated at the mRNA level in many organs and tissues, but at the cellular level it is mainly expressed in osteoclast progenitor cells, osteoclasts, B- and T-lymphocytes, and in dendritic cells (Anderson et al., 1997; Hsu et al., 1999). Regulation of RANK expression has been much less extensively studied than that of RANKL. It has been reported, however, that TGF-ß (Yan et al., 2001) and D3 (Ahlen et al., 2002; Palmqvist et al., 2002) can increase RANK mRNA and that IL-4 (Lerner and Conaway, 2000) as well as activation of gp130 by IL-6 (and the sIL-6R; Palmqvist et al., 2002) can decrease RANK mRNA. The effects of TGF-ß and D3 are in accordance with their osteoclastogenic effects, which also is true for IL-4, a known inhibitor of osteoclast formation. However, the decreased RANK expression caused by gp130 stimulation is surprising when one considers the stimulatory effect on bone resorption by IL-6 (+ sIL-6R). The inhibitory effect of IL-4 on RANK expression in osteoclast progenitor cells is in contrast to the stimulatory effect on T-lymphocytes (Anderson et al., 1997). Recently, we have found that dexamethasone also enhances the mRNA expression of RANK, which may partly explain the potentiation by glucocorticoids on osteoclast formation induced by PTH and D3. Interestingly, neither TNF nor RANKL affects RANK expression, but co-stimulation with both cytokines results in a substantial up-regulation of RANK mRNA (Zhang et al., 2001), which is likely to be one mechanism by which TNF synergistically potentiates the osteoclastogenic effect of RANKL (Abu-Amer et al., 2000; Lam et al., 2000; Zhang et al., 2001). The neuropeptides VIP and PACAP-38 have been shown to decrease D3-stimulated RANK mRNA, which might, at least partly, explain the inhibitory effects of these neuropeptides on osteoclastogenesis (Mukohyama et al., 2000).
Similar to RANKL-deficient mice, RANK knockout mice exhibit an osteopetrotic phenotype due to the absence of multinucleated osteoclasts (Dougall et al., 1999; J Li et al., 2000). The bone marrow cavities are occluded and filled with cartilage encased in mineralized bone matrix. Similar to RANKL–/– mice, the mice deficient in RANK exhibit widened and disturbed organization of the growth plate. The osteopetrotic phenotype of RANK–/– mice, in contrast to that of RANKL–/– mice, can be prevented by bone marrow transplantation, demonstrating the intrinsic defect in the osteoclastic lineage of RANK–/– mice. This is also demonstrated by the fact that no osteoclasts are formed when spleen cells are incubated with M-CSF and RANKL. The absence of osteoclasts in RANK–/– mice is not due to a lack of myeloid or progenitor stem cells, since differentiation and function of macrophages and dendritic cells from their myeloid precursors are normal. The RANK-deficient mice exhibit increased extramedullary hematopoiesis in the spleen, but not in the liver, and a deficiency of B-cells in the spleen. The teeth in RANK–/– mice are smaller, with apparently normal enamel, dentin, cementum, and odontoblasts, but fail to erupt, the latter being a typical finding in mice with decreased osteoclast differentiation or function. RANK deficiency also results in hypocalcemia, hypophosphatemia, and elevated serum levels of PTH. These mice are also resistant to the hypercalcemic response and to the increase of osteoclast number induced by injection of PTHrP, D3, or IL-1ß. Nor does TNF- cause any hypercalcemia. Interestingly, however, osteoclasts are formed in the vicinity of the site of TNF- injection, suggesting that TNF- signaling can partly compensate for the lack of RANKL-RANK interaction. The RANK–/– mice have normal intestinal lymphoid tissues, including Peyer’s patches, but completely lack all other peripheral lymph nodes. Thus, the phenotype of RANK–/– mice is identical to that of RANKL–/– mice, with the exception that thymic differentiation is intact in RANK–/– mice but impaired in RANKL–/– mice.
| (6) Osteoprotegerin |
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Secreted OPG acts as a ‘decoy receptor’ due to its affinity to both membrane-bound and soluble RANKL and prevents the activation of RANK. Because OPG is present as a homodimer and because of the low degree of sequence homology between RANK and OPG, it has been questioned if OPG binds to RANKL in a manner similar to the trimeric interaction between RANK and RANKL. OPG can also bind to TRAIL, a member of the TNF ligand superfamily, although the affinity between OPG and TRAIL is considerably less than that between OPG and RANKL.
Because of its ‘decoy receptor’ function, OPG inhibits osteoclast formation in bone marrow cultures, as well as bone resorption in organ-cultured fetal rat long bones and newborn murine calvariae stimulated by a variety of hormones and cytokines (Kwon et al., 1998; Palmqvist et al., 2002). Injection of OPG into rats or mice results in decreased bone mineral density, bone volume, trabecular bone area, and reduced numbers of osteoclasts (Simonet et al., 1997; Yasuda et al., 1998a). Transgenic mice overexpressing OPG, under the control of the apolipoprotein E gene promoter and its liver-specific enhancer, have a normal appearance, but the bones are osteopetrotic with enhanced bone mineral density, primarily in the trabecular bone, and have decreased numbers of trabecular osteoclasts (Simonet et al., 1997). In contrast to RANKL–/– and RANK–/– mice, the shapes and sizes of bones are normal, and no abnormalities in tooth eruption, lymphocyte development, or thymus and lymph node organogenesis can be found. However, similar to most osteopetrotic animals, OPG transgenic mice exhibit splenomegaly. The decrease of osteoclasts is not associated with any decrease of F4/80-positive monocyte/osteoclast progenitor cells.
Targeted ablation of the OPG gene results in mice exhibiting a substantial decrease of bone density in both trabecular and cortical bone (Bucay et al., 1998; Mizuno et al., 1998). This phenotype is evident early post-natally, and an increased incidence of vertebral and endochondral bone fractures was already noted within the first two weeks. Thus, OPG–/– mice show signs of early-onset osteoporosis. Histological analysis revealed that the bone present in OPG-deficient mice is of mainly a woven type, reflecting decreased remodeling of the skeleton. The decreased bone density is due to enhanced numbers of osteoclasts in the skeleton. The finding that osteoclast formation in spleen cell and bone marrow cultures stimulated by RANKL and M-CSF is similar in OPG–/– mice and wild-type mice indicates that the intrinsic defect is not confined to the number of osteoclast progenitor cells and is in agreement with the finding that OPG is expressed by stromal cells/osteoblasts. This view is further supported by the observations that: (i) osteoblasts from OPG–/– mice support osteoclast formation in co-cultures with osteoclast progenitor cells, even in the absence of hormones stimulating osteoclastogenesis; (ii) basal bone resorption in cultured fetal rat long bones is increased when compared with resorption in wild-type mice; and (iii) RANKL mRNA expression in osteoblasts from OPG–/– is not different from that in wild-type mice (Udagawa et al., 2000). Surprisingly, OPG-deficient mice also exhibit calcification in the media of aorta and renal arteries, but not in smaller arteries, veins, or capillaries.
OPG is expressed more widely and to a much higher extent than RANKL. OPG mRNA has been detected in bone, cartilage, aorta, skin, lung, heart, kidney, liver, brain, and in several other tissues. At the cellular level, OPG is expressed in osteoblasts, stromal cells, endothelial cells, aortic smooth-muscle cells, fibroblasts, dendritic cells, and lymphoid cell lines. It is apparent that osteoclast formation and activation are critically regulated by the RANKL-RANK-OPG system and that the relative expression of these molecules will determine the numbers of osteoclasts formed and consequently the bone mineral density of the skeleton. Very potent stimulators of osteoclastogenesis and bone resorption, such as PTH and D3, substantially increase the ratio RANKL/OPG by increasing RANKL and decreasing OPG expression. At variance, stimulators of bone resorption—such as IL-1, TNF-, TGF-ß, IL-6 (+sIL-6R), IL-11, OSM, and LIF—increase both RANKL and OPG expression, which is the likely explanation for the reduced effectiveness of these cytokines to stimulate bone resorption as compared with PTH and D3. Glucocorticoids, which stimulate bone resorption in organ-cultured bones and synergistically potentiate the osteoclastogenic effects of PTH and D3 in bone marrow cultures, decrease OPG expression in osteoblasts. The suppressing effect of PTH on OPG expression is mimicked by forskolin (Takami et al., 2000) and is at variance with PTH-induced RANKL expression, which is dependent on both the transcription factors CREB and c-fos, but not on Cbfa1 (Fu et al., 2002). Activation of PKC does not mimic the stimulatory effect of PTH but leads to increased OPG expression (Takami et al., 2000).
The relatively restricted phenotype of OPG transgenic and knockout mice is somewhat surprising when one considers the ubiquitous expression of OPG and the fact that RANKL expression is not restricted only to bone (Simonet et al., 1997). This suggests that OPG treatment of pathologically increased bone resorption would be an interesting possibility. Clinical trials are already ongoing for the treatment of post-menopausal osteoporosis and metastatic bone disease.
The importance of OPG has also been investigated by studies of the presence and distribution of polymorphisms in promoter and intron regions of the OPG gene (Langdahl et al., 2002). Twelve different polymorphisms were detected; none of these was associated with changes in bone mineral density or biochemical markers of bone turnover in normal controls. Two of the alleles, A163G and T245C, were significantly more common in osteoporotic patients with vertebral fractures.
| (7) Downstream Intracellular Signaling Molecules |
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B through activation of MAPKs and inhibitory B kinase (IKK), respectively (Fig. 3A). Two groups have generated TRAF6–/– mice and found them to be osteopetrotic and to exhibit defective tooth eruption, B-cell differentiation, lymph node organogenesis, and IL-1 signaling (Lomaga et al., 1999; Naito et al., 1999). In the study by Lomaga et al. (1999), the osteopetrotic mice had normal amounts of TRAP-positive multinucleated osteoclasts, but the osteoclasts lacked contact with bone surfaces, indicating that it was the later stages of osteoclast differentiation, including polarization and activation, rather than differentiation and fusion of osteoclast progenitor cells, that are dependent on TRAF6 expression. This is, to some extent, unexpected, since deficiency of RANK leads to arrest of osteoclast progenitor cell differentiation. Similar to the phenotype of RANK–/– mice, the TRAF6–/– mice generated by Naito et al. (1999) lacked TRAP-positive multinucleated osteoclasts, and the osteoclast progenitor cells do not respond to RANKL/M-CSF stimulation with increased formation of mature osteoclasts. The discrepancy in the two TRAF6–/– models may be due to differences in targeting constructs, indicating the possibility that different domains of TRAF6 might be important for differentiation and maturation of osteoclasts. Transfection of TRAF6–/– spleen cells with wild-type TRAF6 restores the osteoclastogenic response to RANKL/M-CSF (Kobayashi et al., 2001). Using different constructs, Kobayashi et al. (2001) were able to demonstrate that the RING finger of TRAF6 is responsible for maturation of osteoclasts, whereas the second and third zinc fingers are important for differentiation of the osteoclast progenitor cells. Thus, different subdomains of TRAF6 seem to be involved in distinct post-receptor pathways. In line with this view, Kobayashi et al. (2001) demonstrated that the RING finger is necessary for full activation of c-Jun amino-terminal kinase (JNK) and p38 MAPK, but not for NF-B activation. Further support for the view of the presence of different functional domains in the interactions between RANK and TRAFs has been presented by Armstrong et al. (2002), using spleen cells from RANK–/– mice transfected with different RANK constructs selectively incapable of binding different TRAF proteins. It was shown that osteoclast progenitor cell differentiation to multinucleated osteoclasts was not blocked unless all TRAF-binding sites were deleted. In contrast to this functional redundancy in the TRAF pathways downstream from RANK with regard to differentiation, it was demonstrated that TRAF6 is indispensable for the organization of the osteoclast cytoskeleton and the resorptive activity, in line with the observations by Lomaga et al. (1999) in TRAF6–/– mice. The osteopetrotic phenotype of TRAF6–/– mice is not observed in TRAF2- or TRAF5-deficient mice. However, TRAF5–/– mice still exhibit a bone cell phenotype, since osteoclast formation in bone marrow cultures stimulated with M-CSF and either RANKL or TNF- is significantly decreased (Kanazawa et al., 2003). Deletion of the TRAF5 gene also results in a delayed hypercalcemic response in mice injected with PTH.
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). Thus, RANK stimulation causes activation of IKK, which induces a rapid serine phosphorylation of IB, leading to ubiquitination and proteasomal degradation (Wei et al., 2001). This is followed by transactivation of the IB gene and increased cytosolic IB protein. As a consequence of IB phosphorylation, the IB/NF-B complex dissociates, followed by translocation of the NF-B subunits p50/p65 to the nucleus. NF-B consists of homo- or heterodimers of five members of the Rel family, including p50 (NF-B1), p52 (NF-B2), p65 (RelA), RelB, and c-Rel. The importance of the NF-B pathway for osteoclast formation is demonstrated by the finding that mice deficient in both NF-B subunits p50 and p52 are osteopetrotic, with marrow cavities filled with unremodeled osteocartilaginous matrix (Franzoso et al., 1997; Iotsova et al., 1997). The p50/p52–/– mice lack not only mature osteoclasts but also TRAP-positive mononuclear progenitor cells. However, RANK-expressing osteoclast progenitors are present in p50/p52 knockouts (Xing et al., 2002). That it is the lack of p50/p52 expression in osteoclast progenitor cells that is the cause of defective osteoclast formation is indicated by the observations that co-culture of osteoblasts from knockout mice with spleen cells from wild-type mice results in the formation of mature osteoclasts, whereas co-culture of wild-type (or knockout) osteoblasts with spleen cells from p50/p52-deficient mice does not result in the formation of osteoclasts. In line with these observations, the osteopetrotic phenotype of p50/p52–/– mice can be prevented by bone marrow transplantation. Activation of NF-B by RANK is dependent on RANK-induced elevations of intracellular Ca2+ (Komarova et al., 2003). As described above, the transcription factor c-Fos signaling is an important pathway downstream RANK (Grigoriadis et al., 1994). In a genome-wide screening of mRNAs expressed in RANKL-stimulated RAW 264.7 cells (Ishida et al., 2002) and in RANKL-stimulated mouse bone marrow cells (Takayanagi et al., 2002b), it has been shown that the transcription factor NFATc1 (=NFAT2/NFATc) is one of the strongest genes induced. NFATc1 is a member of the nuclear factor of activated T-cells (NFAT) family of transcription factors, which were originally found to be important in regulation of the immune system. Induction of NFATc1 involves RANK-induced Ca2+ oscillations and activation of Ca2+/calmodulin-dependent calcineurin (a serine/threonine phosphatase), which is necessary for translocation of NFATc1 to the nucleus (Takayanagi et al., 2002b). Loss-of-function mutation in the NFATc1 gene leads to abolished capacity to form osteoclasts after RANKL stimulation, whereas M-CSF stimulation of monocyte/macrophage precursors is normal (Takayanagi et al., 2002b). NFATc1 forms a complex with c-Fos, and expression of both transcription factors is necessary for the expression of TRAP, and calcitonin receptors, which are similar to the genes for cathepsin K, carbonic anhydrase II, and MMP-9, contain binding sites for both NFATc1 and AP-1.
Three major subfamilies of MAP kinases have been identified: (i) extracellular signal-regulated kinases (ERKs), (ii) JNK, and (iii) p38 MAP kinase. These can be activated by MAP kinase kinase (MKK)-mediated phosphorylation on threonine and tyrosine residues. The involvement of this pathway in osteoclastogenesis is indicated by the observation that JNK is not activated in TRAF6–/– mice (Lomaga et al., 1999) and by the finding that RANK overexpression leads to enhanced activation of JNK and NF-B (Hsu et al., 1999). Using cells transfected with RANK C-terminal deletion mutants, Hsu et al. (1999) showed that RANK-induced activation of JNK and NF-B correlates with the TRAF6-binding domain of RANK. Matsumoto et al. (2000) have shown that RANKL stimulates phosphorylation of JNK, ERK, and p38 MAP kinase in RAW 264.7 cells, an osteoclast progenitor cell line which can differentiate to mature osteoclasts in the presence of RANKL. Pharmacological inhibitors have been used to show that osteoclastogenesis was associated with p38 MAP kinase, and strong evidence for the crucial role of this kinase was provided by demonstrations that the expression of the dominant-negative form of either p38 MAP kinase or MKK6 inhibited RANKL-induced osteoclast formation. RANKL-induced stimulation of p38 MAPK is involved in the differentiation of osteoclasts, but not in their survival or bone-resorbing activity (Li et al., 2002). Recently, David et al. (2002) have shown, by using JNK1–/– and JNK2–/– mice, that RANKL activates JNK1 preferentially and that lack of JNK1 results in decreased (~ 50%) osteoclast formation. In addition, RANKL stimulation of bone marrow cells from either c-Jun–/– or JunD–/– mice, or RANKL-stimulated bone marrow cultures from mice (JunAA/JunAA) carrying a c-Jun mutant rendering c-Jun less sensitive to phosphorylation by JNK, revealed that JNK-1-dependent phosphorylation of c-Jun, and c-Jun itself, are important for RANKL-induced osteoclastogenesis. Interestingly, JNK1 seems to affect osteoclastogenesis by two mechanisms: (i) JNK1 protects osteoclast progenitor cells from RANKL-induced apoptosis, a mechanism independent of c-Jun phosphorylation; and (ii) a c-Jun phosphorylation-dependent increased differentiation of osteoclast progenitor cells.
Signaling pathways downstream of RANK (similar to that of M-CSF) also include activation of PI3K and of the anti-apoptotic serine/threonine kinase Akt (protein kinase B) (Fig. 3B). Activation of PI3K and Akt by RANK is dependent on TRAF6/c-src interactions (Wong et al., 1999; Arron et al., 2001). The Akt survival signaling pathway is crucial for the anti-apoptotic and osteoclastogenic activity of RANK. Recently, Sugatani et al. (2003) reported that RANK activation of Akt results in phosphorylation of Bad, a member of the Bcl-2 family, which, through phosphorylation of Ser-136 by Akt, becomes inactivated and loses its apoptotic activity. The PI3K/Akt/Bad pathway is inhibited by the tumor suppressor gene PTEN (phosphatase and tensin homologue deleted from chromosome 10; Sugatani et al., 2003). PI3K generates the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3], which can be dephosphorylated by PTEN to the inactive PtdIns(4,5)P2. PTEN also can act as a protein phosphatase by dephosphorylating active Akt. Thus, PTEN can inhibit the PI3K/Akt pathway by two mechanisms. PTEN overexpression suppresses RANKL-stimulated differentiation of RAW 264.7 cells to osteoclasts, whereas transfection with dominant-negative PTEN increases osteoclast formation. Although the data cannot fully discriminate between effects of PTEN on osteoclast number as being a consequence of effects on differentiation or on cell survival/apoptosis, the most likely explanation is that PTEN interferes with the PI3K/Akt/Bad signaling involved in RANK-mediated anti-apoptosis. Sugatani et al. (2003) also provide evidence that RANK may regulate the expression of PTEN in osteoclasts, which suggests that PTEN plays a role in regulating the balance between active and non-active Akt. Interestingly, inhibition of PI3K by PTEN also inhibits osteopontin-stimulated migration of RAW 264.7 cells, by a mechanism unrelated to Akt/Bad signaling. Besides PTEN [dephosphorylating PtdIns(3,4,5)P3 by acting as a 3'-phosphatase], SHIP and SHIP2 can also contribute to the dephosphorylation of PtdIns(3,4,5)P3 by acting as 5'-phosphatases. SHIP is a hematopoietic-restricted Src homology (SH) 2-containing inositol-5-phosphatase (SHIP), which is tyrosine-phosphorylated by various cytokines, including M-CSF. Mice deficient in SHIP have increased numbers of osteoclasts due to the increased life span of these cells and to increased sensitivity to the osteoclastogenic effects of M-CSF and RANKL (Takeshita et al., 2002). SHIP–/– osteoclasts are enlarged and contain huge numbers of nuclei. Due to the large numbers of giant osteoclasts, mice deficient in SHIP are osteoporotic with decreased bone mass and reduced trabecular thickness, number, and connectivity. Thus, inhibition of the PI3K pathway, by either PTEN or SHIP, seems to be a negative regulator of osteoclast formation. The phenotype of SHIP–/– mice, including large osteoclasts containing a huge number of nuclei, increased serum levels of IL-6, and hypersensitivity to RANKL, is very similar to that of patients with Paget’s disease.
| (8) RANKL-RANK-OPG in Dental Tissues |
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The crucial role of the RANKL-RANK-OPG system in tooth eruption is most obvious by the observations that mice deficient in RANKL or RANK (or some of the downstream signaling molecules) do not have erupted teeth. For an extensive review of the molecular mechanisms involved in tooth eruption, the reader is referred to Wise et al. (2002b).
The RANKL-RANK-OPG system is also involved in the bone resorption taking place during orthodontic tooth movement. In wild-type mice, osteoclasts are formed at the pressure site in the periodontal ligament and in adjacent alveolar bone. In OPG–/– mice, the number of osteoclasts formed increases dramatically, leading to extensive resorption and perforation of alveolar bone as early as at 2 and 5 days after force application (Oshiro et al., 2002).
| (9) RANKL/RANK/OPG System in Pathological Conditions |
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