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Osteoclasts are highly specialized cells capable of degrading mineralized tissue and form at different regions of bone to meet different physiological needs, such as mobilization of calcium, modeling of bone structure, and remodeling of bone matrix. Osteoclast production is elevated in a number of pathological conditions, many of which lead to loss of bone mass. Whether normal or pathological, osteoclastogenesis strictly depends upon support from accessory cells which supply cytokines required for osteoclast differentiation. Only one of these cytokines, receptor activator of NFκB ligand (RANKL), is absolutely essential for osteoclast formation throughout life and is thus expressed by all cell types that support osteoclast differentiation. The central role of RANKL in bone resorption is highlighted by the fact that it is the basis for a new therapy to inhibit bone loss. This review will discuss mechanisms that control RANKL gene expression in different osteoclast-support cells and how the study of such mechanisms may lead to a better understanding of the cellular interactions that drive normal and pathological bone resorption.
It has been more than a decade since the cytokine known as receptor activator of NFκB ligand (RANKL) was shown to be essential for osteoclast differentiation in mice . Prior to that discovery, osteoclast differentiation in vitro could only be accomplished by incubating osteoclast precursors, consisting of hematopoietic cells from bone marrow or spleen, with support cells, usually primary calvaria cells or bone marrow stromal cells [2, 3]. In most cases, these co-cultures also required the addition of a factor known to stimulate bone resorption in vivo, such as parathyroid hormone (PTH) or 1,25 dihydroxyvitamin D3 (1,25(OH)2D3). We now know that the support cells supply two factors essential for osteoclast formation. One is RANKL, which initiates the osteoclast differentiation pathway and then promotes the survival and activity of differentiated cells . The other is macrophage colony stimulating factor (M-CSF), which promotes osteoclast precursor proliferation as well as the survival and motility of mature osteoclasts [5-7]. All of the actions of RANKL are opposed by a soluble decoy receptor known as osteoprotegerin (OPG) . Concurrent with the discovery of the roles of RANKL and OPG in osteoclast formation, it was found that the hormones and cytokines that stimulate osteoclast formation in co-cultures do so primarily by stimulating RANKL expression, and, in some cases, also suppressing OPG expression .
Although much has been learned about the differentiation pathways activated by RANKL in osteoclast precursors, comparatively less is known about the molecular mechanisms by which various stimuli control RANKL expression in cells that support osteoclast differentiation. Numerous cell types have been reported to express RANKL mRNA or protein. A non-exhaustive list includes both T and B lymphocytes [9, 10], mammary epithelial cells , keratinocytes , vascular endothelial cells , synovial fibroblasts , cells within periodontal tissue , hypertrophic chondrocytes , as well as osteoblast precursors, mature osteoblasts, and osteocytes [16, 17]. Malignant cell types that occur in prostate cancer and multiple myeloma have also been shown to express RANKL [18, 19]. RANKL-deficient mice display defects in lymphocyte maturation and mammary gland differentiation demonstrating the functional significance of expression in lymphocytes and mammary epithelia, respectively [1, 11]. And although RANKL-deficient mice, and humans , clearly lack osteoclasts, it has been somewhat problematic to conclusively identify the cell types that express the RANKL that is responsible for osteoclast differentiation, whether that differentiation occurs during physiological bone remodeling or in pathological conditions. The goal of this review is to summarize current understanding of the mechanisms that control RANKL gene expression in various cell types that are likely to support osteoclast differentiation and how such information may lead to a better understanding of the cellular and molecular underpinnings of bone remodeling.
RANKL is a member of the tumor necrosis factor (TNF) family of cytokines and has been given the specific designation TNF (ligand) superfamily, member 11 (TNFSF11). A RANKL homologue has been identified in species ranging from humans to bony fish (Figure 1). In mammals, the gene structure is highly conserved, consisting of 5 exons that span 33.9 kb in humans and 30.5 kb in mice. The only avian RANKL gene sequenced thus far also contains 5 exons and is somewhat smaller, spanning 22.7 kb. The RANKL homologue in teleost fish is significantly smaller than other vertebrates, consisting of only 4 exons spanning 3.5 kb. At present it is unclear whether a RANKL homologue is present in organisms that lack osteoclasts. In each species with an identifiable RANKL gene, the gene for A kinase anchor protein 11 (AKAP11) is immediately upstream from RANKL and is transcribed from the same strand of DNA, while the gene downstream from RANKL varies. As will be discussed in more detail below, transcriptional control of RANKL in mammals involves regulatory elements that reside more than 70 kb upstream from the first exon. Thus, while the functional significance of the association with AKAP11 is unclear, the maintenance of the extensive intergenic region upstream from RANKL in land-dwelling vertebrates may reflect the complex transcriptional regulatory mechanisms that control RANKL expression in these species.
RANKL is produced in a membrane-bound and soluble form and both are capable of stimulating osteoclast differentiation in vitro [4, 21]. In vitro over-expression studies demonstrate that the soluble form can be generated by proteolytic cleavage of the membrane-bound form by proteinases, such as TNF-α converting enzyme (TACE) , a disintegrin and metalloproteinase (ADAM) 10 , and matrix metalloproteinase (MMP) 14 . However, suppression or genetic deletion of endogenous MMP14 is sufficient to significantly reduce soluble RANKL levels in vitro and in vivo suggesting that this enzyme plays a predominant role in RANKL shedding . In addition to proteolytic cleavage, a soluble form of RANKL can also be produced in human cells by alternative splicing. Specifically, the human gene contains an alternative set of upstream exons that produce a transcript that does not encode a trans-membrane domain, and this transcript may be preferentially produced in malignant cell types . As yet, a transcript encoding a soluble form of RANKL has not been identified in the mouse.
The existence of soluble forms of RANKL suggests the possibility that expression at or near the site of osteoclast differentiation may not be necessary. However, in co-culture systems, cell-to-cell contact between stromal support cells and osteoclast precursors is required for osteoclastogenesis . Consistent with this, shedding of RANKL from stromal cells inhibits osteoclastogenesis in vitro and mice with reduced RANKL shedding have increased osteoclast number . RANKL-deficient mice harboring a transgene expressing RANKL specifically in lymphocytes can form a bone marrow cavity, but this is devoid of trabeculae, highlighting the importance of local expression of RANKL to achieve normal osteoclast formation and function . Moreover, tooth eruption and modeling of the metaphyseal cortex do not occur in these mice demonstrating that regulated expression of membrane-bound RANKL in the appropriate cell type is essential for osteoclastogenesis in at least some areas of the skeleton.
Circulating soluble RANKL levels are strikingly elevated in mice and humans lacking OPG [26-28]. This increase in circulating protein occurs without changes in RANKL mRNA expression, suggesting that OPG may suppress the shedding of RANKL [26, 27]. However, the significance of this action of OPG to its overall role in suppressing bone resorption is unclear since the relationship of endogenous soluble RANKL to osteoclast formation at different bone sites has yet to be established.
Although a number of different cell types have been shown to express RANKL, this section will focus on the mechanisms that control RANKL expression in cell types for which multiple studies suggest involvement in osteoclast formation. Such cell types include stromal or osteoblast lineage cells, chondrocytes, lymphocytes, and endothelial cells.
Extensive circumstantial evidence suggests that cells of the osteoblast lineage are an important source of the RANKL that stimulates osteoclast formation. This evidence consists mainly of localization of RANKL-expressing cells in bone sections [16, 17, 29] and detection of RANKL expression in cells isolated from rodent calvaria, which are rich in osteoblast progenitors [4, 8]. However, these localization studies have provided inconsistent results. For example, some studies detected RANKL in osteocytes while others did not. Moreover, calvarial preparations are heterogeneous and contain other cell types in addition to osteoblast precursors [30, 31], some of which may be sources of RANKL. More importantly, conditional ablation of osteocalcin-expressing cells in mice did not alter osteoclast number or function , demonstrating that mature osteoblasts and their immediate precursors cannot be an essential source of RANKL in bone. Thus while osteoblast precursors may be an important source of RANKL, this has yet to be demonstrated experimentally. This uncertain relationship between osteoclast-support cells, defined by the expression of RANKL, and cells of the osteoblast lineage, defined here as cells that express relatively osteoblast-specific genes such as osteocalcin, has led to the use of vague terms to describe such support cells, such as stromal or stromal/osteoblastic, and it is the latter term that will be used here.
Parathyroid hormone (PTH) is one of the most potent stimulators of RANKL expression in stromal/osteoblastic cells . The concomitant reduction in RANKL levels and osteoclast number in rodents lacking PTH suggests that this regulation is biologically relevant [33, 34]. PTH stimulation of RANKL expression occurs primarily through activation of the protein kinase A (PKA) - cAMP pathway [35-37] and requires the well-known target of this pathway, cAMP response element binding protein (CREB)  (Figure 2). CREB is also required for stimulation of RANKL by 1,25(OH)2D3 and the gp130 cytokine oncostatin M (OSM), suggesting that this transcription factor may play a central role in coordinating the actions of multiple signaling pathways . A potential CREB binding site has been identified 962 bp upstream from the transcription start site of the murine RANKL gene . However, additional studies have not detected significant stimulation of transcriptional reporter constructs containing up to 2 kb of the murine 5′-flanking region by PTH, 1,25(OH)2D3 or OSM [39-41]. This latter set of results suggested that important transcriptional regulatory regions might reside outside the proximal 5′-flanking region of this gene.
To identify potential distant regulatory regions, my laboratory developed an approach in which bacterial artificial chromosomes (BACs) are used to create transcriptional reporter constructs for the RANKL gene. BAC clones can harbor DNA fragments up to 200 kb in length and can be propagated in the same manner as plasmids. Because they contain such large segments of DNA, BAC-based reporter constructs allow regulatory studies to be performed with genes in a more native structure compared to traditional promoter-reporter constructs [42-44]. In many cases, DNA fragments containing an entire gene, spanning tens of kb, can be obtained. Quite often these large fragments also contain the naturally occurring insulators that define the borders between genes . Thus these fragments frequently exhibit copy number-dependent, position-independent expression when used to generate stable cell lines or transgenic animals [42, 46]. In addition, they often contain the necessary regulatory elements to confer appropriate cell type-specificity and responsiveness to extracellular signals [43, 46-50].
The initial BAC-based RANKL reporter construct consisted of the full-length murine RANKL gene, as well as extensive 5′- and 3′-flanking region, in which the 3′-untranslated region was replaced with the luciferase coding sequence using recombineering techniques . In contrast to reporter constructs containing only the proximal RANKL 5′-flanking region, the resulting BAC-based construct was robustly stimulated by PTH, as well as by 1,25(OH)2D3 or OSM . The recombineering approach was then used to delete various regions within the BAC-based reporter construct and thereby localize the PTH-responsive region to a 2 kb fragment located 76 kb upstream from the transcription start-site. Sequences within this 2 kb fragment are highly conserved among mammals and contain a motif harboring two highly conserved cAMP-response elements (CREs) as well as a binding site for the osteoblast-specific transcription factor Runx2 . CREB and Runx2 were shown to bind these sites using gel mobility shift and chromatin immunoprecipitation (ChIP) assays , suggesting that these sites are functional.
To determine the importance of this 2 kb enhancer for control of the endogenous RANKL gene, we have generated mice lacking this region. Deletion of the enhancer, designated the RANKL distal control region (DCR), increased bone mass in both the axial and appendicular skeleton . Loss of the enhancer also reduced PTH stimulation of RANKL mRNA in primary bone marrow cultures, as well as stimulation of RANKL mRNA in bone . DCR-deficient mice also had reduced basal RANKL mRNA levels in bone, thymus, and spleen. The increase in bone mass was due to reduced osteoclast and osteoblast formation leading to a low rate of bone remodeling, similar to that observed in humans and mice with hypoparathyroidism. These findings demonstrate that control of RANKL expression via the DCR is a critical determinant of the rate of bone remodeling. Subsequent studies indicate that mice lacking the DCR are also completely protected from the loss of cancellous bone that occurs with secondary hyperparathyroidism (C. Galli and C.A. O'Brien, unpublished results). Interestingly, cortical bone loss in this model is unaffected by deletion of the DCR, suggesting that different transcriptional enhancers of the RANKL gene mediate the response to secondary hyperparathyroidism in different skeletal compartments.
1,25(OH)2D3 is another potent inducer of RANKL expression in stromal/osteoblastic cells . In addition to its effect on PTH action, deletion of the DCR also reduced the response to 1,25(OH)2D3 in vitro and in vivo [41, 52]. The DCR was independently identified as a potential 1,25(OH)2D3-responsive region in studies by Pike and colleagues that utilized a combined ChIP-DNA microarray approach . These studies identified functional vitamin D response elements (VDREs) within the DCR as well as several additional VDREs within other conserved regions that lie 16, 22, 60, and 69 kb upstream from the transcription start site of the murine gene. Despite the high level of sequence conservation between species, conventional promoter-reporter studies suggest that in the human RANKL gene, one of these more proximal enhancers may play a dominant role in mediating the response to 1,25(OH)2D3 . Several earlier studies had identified a potential VDRE located approximately 1 kb upstream from the transcription start site [55-57]. However, as neither the BAC-based mapping approach nor the ChIP-DNA microarray approach identified this region as mediating effects of 1,25(OH)2D3, the significance of this proximal site to the overall control of the RANKL gene remains to be determined.
Cytokines that utilize the gp130 signal transducer, also known as IL-6-type cytokines, stimulate RANKL in stromal/osteoblastic cells [8, 58]. Members of this family play roles in both normal and pathological bone resorption [59, 60]. Cytokines that utilize gp130 activate the signal transducers and activators of transcription (STAT) and mitogen-activated protein kinase (MAPK) pathways. Studies using dominant-negative proteins demonstrated that STAT3 is essential for stimulation of RANKL by gp130 cytokines and that blockade of gp130 or STAT3 blunted the response to IL-1, suggesting that IL-1 stimulates RANKL indirectly by stimulating the expression of IL-6-type cytokines . This latter finding may also be relevant to the actions of TNFα, as IL-1 has been shown to be required for the actions of TNFα on RANKL expression in stromal/osteoblastic cells . BAC-based reporter assays indicated that an important response element mediating the effects of gp130 cytokines lies within a 10 kb fragment beginning 82 kb upstream from the transcription start site ; however, the sequences within this region that bind STAT3 have yet to be identified.
Compelling evidence indicates that canonical Wnt signaling stimulates OPG expression in bone and thereby suppresses bone resorption [62, 63]. A growing number of studies suggest that the same pathway may also be an important regulator of RANKL in stromal/osteoblastic cells. In vitro, canonical Wnt signaling or over-expression of β-catenin, a transcriptional co-factor that mediates the effects of the canonical Wnt pathway, suppressed RANKL expression, either under basal conditions or after stimulation by PTH or 1,25(OH)2D3 [64-66]. Conversely, deletion of β-catenin from calvarial cultures stimulated RANKL expression . The T cell factor (TCF) family of transcription factors function together with β-catenin to mediate the transcriptional effects of canonical Wnt signaling . Spencer et al. identified several potential TCF binding sites within the human RANKL 5′-flanking region that may mediate the effects of Wnt signaling and over-expression of β-catenin was able to suppress the activity of a murine RANKL promoter-reporter construct containing many of these sites . Several in vivo studies also suggest that canonical Wnt signaling suppresses RANKL expression. Administration of DKK1, which blunts canonical Wnt signaling, stimulated RANKL expression and osteoclast formation in bone  and suppression of DKK1 in ovariectomized rats had the opposite effect . Moreover, in mice with a hypomorphic LRP6 allele, canonical Wnt signaling in bone is decreased and this is associated with increased bone resorption and RANKL expression . Taken together, these studies suggest that canonical Wnt signaling in bone exerts a tonic negative control of RANKL expression. The signaling pathways, transcription factors, and regulatory regions that control RANKL expression in stromal/osteoblastic cells are summarized in figure 2.
The junction between the calcified cartilage at the base of the growth plate and newly formed bone is a site of some of the most robust osteoclast formation within the growing skeleton (Figure 3). These osteoclasts are responsible for initiating the remodeling of the calcified cartilage into cancellous bone. A likely source of the RANKL stimulating the formation of these osteoclasts is the hypertrophic chondrocytes buried within the mineralized matrix. Concordant with this, immunolocalization and in situ hybridization studies consistently detect RANKL expression in hypertrophic chondrocytes [4, 16, 29, 72, 73]. Deletion of the vitamin D receptor in chondrocytes reduced RANKL expression and osteoclast formation in young mice, suggesting that 1,25(OH)2D3 may be an important regulator of RANKL expression in these cells, in addition to its role in stromal/osteoblastic cells . In vitro studies have demonstrated that BMPs are also able to stimulate RANKL expression, as well as a RANKL promoter-reporter construct, in isolated chondrocytes . Stimulation of the reporter construct, which contained less than 1 kb of the murine 5′-flanking region, required Runx2 binding sites within the proximal promoter suggesting that BMP-activated SMADs may function together with Runx2 to control RANKL expression in this cell type. As was the case with hormonal control, it will be important to determine whether more distant enhancers contribute to the effects of BMPs on RANKL transcription.
RANKL was originally identified as a TNF family member highly expressed in T lymphocytes [9, 76]. Shortly thereafter, several studies demonstrated that activated T and B cells can support osteoclast formation in vitro via expression of RANKL [77-79], suggesting that these cell types may be important sources of RANKL during inflammation. Subsequently, studies in humans demonstrated that RANKL protein levels on B and T lymphocytes isolated from the bone marrow of post-menopausal women were elevated compared with pre-menopausal women or post-menopausal women treated with estrogen , suggesting that lymphocyte expression of RANKL may also play an important role in the elevated bone resorption associated with loss of sex steroids. It should be noted, however, that there are as yet no studies demonstrating direct control of RANKL gene transcription by sex steroids in any cell type.
The first evidence that lymphocyte activation can play a role in stimulating osteoclast formation in vivo was obtained by examining mice lacking the gene cytotoxic T-lymphocyte-associated protein 4 (ctla4). Ctla4 is expressed on the surface of T cells and acts as a homeostatic suppressor of their activation and mice lacking this gene exhibit constitutive T cell activation . This increase in T cell activation was found to be associated with increased osteoclast number and reduced bone mass, which were reversed by administration of OPG . Based on this result, the authors of the study concluded that elevated expression of RANKL on activated T cells is sufficient to increase bone resorption. However, a more recent study has shown that ctla4 directly inhibits osteoclast differentiation in the presence of constant amounts of RANKL , raising the possibility that the increased osteoclastogenesis in ctla4-deficient mice may not involve RANKL expression by activated T cells.
It is important to note that administration of OPG inhibits osteoclast formation and bone resorption in all in vivo models because osteoclast differentiation in vivo is absolutely dependent on RANKL expression . Therefore, blockade of bone resorption in a particular model by addition of exogenous OPG cannot reveal whether changes in RANKL expression are responsible for an increase in bone resorption. Moreover, elevation of many other cytokines such as M-CSF , IL-1 , and TNFα  can synergize with constant levels of RANKL to increase osteoclast differentiation. Thus, the ability of OPG to reduce osteoclast numbers in ctla4-deficient mice does not reveal whether the increase in osteoclasts was due to elevated RANKL expression on T cells or any other cell type.
Although it is clear that activated T cells can stimulate osteoclast formation in vitro, a seminal study by Hiroshi Takayanagi demonstrated that isolated live T cells, activated by anti-CD3 antibody, completely inhibited osteoclast formation in vitro . The mechanism for this inhibition involved production of IFNγ by the T cells which promoted degradation of TRAF6, a scaffolding protein essential for osteoclast differentiation, in osteoclast precursors. Thus the ability of activated T cells to stimulate or inhibit osteoclast differentiation in vitro depends on the method of activation and type of culture system used. Perhaps more importantly, inflammation due to lipopolysaccharide injection, collagen-induced arthritis, or serum transfer-induced arthritis, can induce bone loss in T cell-deficient mice [86-88]. Thus, T cells cannot be the sole source of RANKL in inflammatory bone loss. Nonetheless, depletion or genetic deletion of T cells significantly blunts bone loss in some murine models of inflammation  or estrogen deficiency . However, in inflammatory models, lack of T cells also blunts the inflammatory response in general. Therefore, it remains unclear whether the role of T cells in inflammation-associated bone loss is to express RANKL, to promote the inflammatory response, or both.
At the transcriptional level, little is known about the control of RANKL expression in lymphocytes. Only a single study has examined the activity of RANKL promoter-reporter constructs in a T cell model . This study demonstrated that RANKL promoter-luciferase constructs, containing approximately 2 kb of the proximal 5′-flanking region, were stimulated by phorbol-12-myristate-13-acetate (PMA) and ionomycin in the human Jurkat T cell line. The combination of PMA and ionomycin treatment, which mimics activation of the T cell receptor, increased binding of NFκB and Egr family transcription factors to gel shift probes corresponding to putative sites in the RANKL proximal promoter region . However, the requirement of these binding sites for promoter activity was not determined.
The decrease in RANKL expression observed in the thymus and spleen of mice lacking the DCR enhancer may reflect reduced expression in T and B cells, respectively. If this is found to be the case, it will be important to determine whether the same transcription factor binding sites that mediate DCR activity in stromal/osteoblastic cells, such as the CREs, also play a role in lymphocytes.
Another potential cellular source of RANKL that may be involved in osteoclast formation is vascular endothelial cells. The close anatomical association of blood vessels with osteoclasts has been repeatedly observed  and suggests that the progenitor cells required for the advancing basic multicellular unit (BMU) are supplied by such vessels, although a functional relationship between neo-vascularization and osteoclast formation has yet to be established. Nonetheless, Collin-Osdoby and colleagues have reported that the inflammatory cytokines IL-1 and TNFα stimulated RANKL expression in human microvascular endothelial cells at levels sufficient to promote osteoclast formation in vitro , suggesting that RANKL expression in the vasculature plays an important role in the osteoclast formation and bone resorption associated with inflammation. Others have shown that TGFβ stimulates RANKL in murine bone marrow-derived endothelial cells via p38 MAP kinase and subsequent activation of CREB-family transcription factors . This latter study also found that TGFβ stimulated activity of a murine RANKL promoter-reporter construct containing approximately 1 kb of 5′-flanking region, although the specific sequences involved were not identified. Because TGFβ effects on bone resorption are complex, in many cases inhibiting osteoclast formation , the significance of its control of RANKL in endothelial cells remains unclear.
Although multiple cells types may supply the RANKL that is required for osteoclast formation, much attention has been focused on stromal/osteoblastic cells because expression of RANKL in this cell type may help to explain a fundamental aspect of bone remodeling. During physiological bone remodeling, and even in many pathological conditions, osteoblasts form bone only at sites previously occupied by osteoclasts. Several mechanisms have been proposed to explain this phenomenon, which is often referred to as coupling . One potential explanation for coupling stems from the observation that osteoblast lineage cells mediate the osteoclastogenic actions of hormones that stimulate bone resorption. This observation led to the suggestion, almost 30 years ago, that osteoblast lineage cells are required for osteoclast formation . We now know that most hormones and cytokines that stimulate osteoclast formation do so by stimulating expression of RANKL on stromal cells. However, as mentioned earlier, the exact relationship of such stromal cells to cells of the osteoblastic lineage is unclear. Nonetheless, if osteoblast lineage cells are indeed an essential source of RANKL for osteoclast development, such a requirement may contribute to the coupling of bone formation to bone resorption. Specifically, in this scenario, osteoclast formation could not take place unless precursors of the cells necessary to replace the bone removed by osteoclasts were anatomically nearby. Put another way, linking osteoclast formation to osteoblast formation would ensure that osteoblast progenitors are locally available to supply the osteoblasts needed to follow the osteoclasts in the remodeling cycle. Clearly additional mechanisms would also be required to promote migration and differentiation of the osteoblast progenitors.
Since, at present, there is only circumstantial evidence suggesting that osteoblast lineage cells are required for osteoclast formation, one goal of our laboratory has been to obtain molecular and genetic evidence to support or refute such a linkage. Initially these studies focused on identification of the molecular mechanisms that control RANKL expression in mesenchymal cells derived from murine bone marrow or calvaria since such cells appeared most likely to be involved in the support of osteoclast differentiation. In the course of these studies, we identified highly conserved Runx2 binding sites in the proximal 5′-flanking region of the RANKL gene as well as in the DCR [41, 96]. Since each of these sites has been shown to bind Runx2, and since this factor is absolutely essential for osteoblast formation [97, 98], it would be reasonable to speculate that Runx2 control of RANKL expression provides a molecular basis for a linkage between osteoblast and osteoclast formation.
Despite these consistent findings, subsequent studies have not been able to demonstrate a functional role for Runx2 in RANKL expression in stromal/osteoblastic cells. Specifically, expression of a dominant-negative form of Runx2 in a stromal/osteoblastic cell line had no effect on RANKL expression . Moreover, even though osteoclasts are reduced in Runx2-deficient mice, cell lines derived from these mice express RANKL in response to 1,25(OH)2D3 [38, 99]. Consistent with this, mouse embryonic fibroblasts (MEFs) from Runx2-deficient mice and wild type littermates express equivalent amounts of RANKL in response to activation of the cAMP-PKA pathway . In each of these studies, it remained possible that other members of the Runx family of transcription factors compensated for the loss of Runx2 since each member of the family can bind to the same DNA sequences. To address this issue, short hairpin RNAs were used to suppress expression of Cbfb, a Runx family binding partner essential for their activity, in several mesenchymal cell models that express RANKL. Although knock-down of Cbfb significantly reduced expression of known Runx target genes, it had no effect on basal or stimulated RANKL expression . Thus in the cell models tested, which includes stromal/osteoblastic cell lines, primary calvaria cells, and MEFs, Runx2 is not required for expression of RANKL, either under basal conditions or in response to hormonal stimuli.
The evidence that Runx2 is not required for RANKL gene transcription in mesenchymal cells does not necessarily lead to the conclusion that osteoblast lineage cells are an unimportant source of RANKL in vivo. For example, it remains possible that osteoblast-lineage cells are an essential source of RANKL, not because cell type-specific transcription factors limit RANKL transcription to this cell type, but rather, because osteoblast-lineage cells can preferentially respond to hormones or other stimuli that activate pathways able to stimulate RANKL transcription. Consistent with this idea, PTH-induced RANKL was higher in cells expressing osterix, a transcription factor highly expressed in osteoblast-lineage cells, and this was associated with higher levels of the PTH receptor in the osterix-expressing cells . Importantly, use of dibutyryl-cAMP to by-pass the PTH receptor induced equivalent amounts of RANKL in osterix-positive and osterix-negative cell populations. Similarly, Yamamoto et al. demonstrated that BMP-2 treatment of primary muscle cells increased PTHR1 expression along with the ability of PTH to stimulate RANKL in these cells . Thus, cell type-specific expression of receptors or signaling pathway components may play a role in limiting RANKL expression to stromal/osteoblastic cells in vivo.
Over the past decade, several signaling pathways and transcription factors that control RANKL gene expression have been identified, primarily those activated by osteoclastogenic hormones such as PTH and 1,25(OH)2D3 in stromal/osteoblastic cells. These studies, together with analysis of the cis-acting elements that mediate responses to these hormones, have revealed unexpectedly complex regulatory mechanisms. Such mechanisms involve multiple transcriptional enhancers, some of which are quite distant from the transcription start site. It is interesting to note that some of the most significant single nucleotide polymorphisms (SNPs) associated with bone mass in humans are within a haplotype block upstream from the RANKL gene that includes the distant regulatory elements . The BAC-based reporter approach used to identify the distant enhancers may prove useful for testing the functional impact of these polymorphisms on RANKL expression.
The presence of multiple transcriptional enhancers, spaced over large distances upstream from the RANKL gene, may reflect the essential role of RANKL in the control of bone resorption. Specifically, since no other gene can compensate for loss of RANKL in vivo, it is possible that some of these enhancers serve redundant functions so that spontaneous mutations affecting just one of them would be insufficient to cripple osteoclast formation. Albeit, the requirement of the DCR to achieve normal osteoclast numbers in cancellous bone indicates a lack of redundancy for at least some RANKL enhancers. The presence of redundant enhancers may also have played an important role in the development of RANKL expression in different cell types and in response to different stimuli. Behringer and colleagues have suggested that redundancy of transcriptional enhancers may promote the evolution of changes in expression by providing a substrate for mutational changes that will not disrupt existing expression . It is also possible that multiple enhancers are needed to control RANKL expression in osteoclast support cells at different skeletal sites under different conditions. For example, the osteoclast formation required for modeling of the periosteal surface of the metaphasis during long bone growth is likely to be controlled independently of the osteoclast formation that drives remodeling of cancellous bone (Figure 3). Determination of how known enhancers, such as the DCR, function, as well as identification and deletion of additional enhancers, will be required to address these issues.
For these studies to provide meaningful results, it will also be important to definitively identify the cellular sources of RANKL that are essential for osteoclast formation. A powerful approach to help resolve this issue will be to delete the RANKL gene in specific cell populations using Cre-loxP technology. Mice which begin expressing the Cre recombinase at various stages of osteoblast differentiation are available and their use, in conjunction with a conditional RANKL allele, should help define the stages at which RANKL expression may be important. Likewise, mice expressing Cre recombinase in chondrocytes, T or B lymphocytes, and vascular endothelial cells are also available and will help to clarify the role of RANKL expression in these cell types under both basal and pathological conditions.
The author would like to thank Robert L. Jilka, Robert S. Weinstein, and Stavros C. Manolagas for critical reading of the manuscript and Jinhu Xiong for preparation of the histological section used in figure 3. This work was supported by the NIH (AR049794 and AG139181) and the Department of Veterans Affairs (Merit Review).
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