Proteins
In addition to the two key osteoclastogenic cytokines M‐CSF and RANKL, a number of other proteins play important roles in osteoclast biology, either in physio- logical and/or pathophysiological circumstances.
As discussed earlier, OPG, a high‐affinity ligand for RANKL that acts as a soluble inhibitor of RANKL, is secreted by cells of mesenchymal origin, both basally and in response to other regulatory signals, including cytokines and bone‐targeting steroids [2,26]. Proinflammatory cytokines suppress OPG expression while simultaneously enhancing that of RANKL, with the net effect being a marked increase in osteoclast formation and function.
Genetic deletion of OPG in both mice and humans leads to profound osteoporosis, whereas overexpression of the molecule under the control of a hepatic promoter results in severe osteopetrosis [6,26]. Together, these observa- tions indicate that skeletal and perhaps circulating OPG modulates the bone resorptive activity of RANKL and helps to explain the increased bone loss in clinical situa- tions accompanied by increased levels of TNF, IL‐1, PTH, or PTH‐related protein (PTHrP). Serum PTH levels are increased in hyperparathyroidism of whatever etiology, whereas PTHrP is secreted by metastatic lung and breast carcinoma [27,28]. TNF antibodies or a soluble TNF recep- tor‐IgG fusion protein potently suppress the bone loss in disorders of inflammatory osteolysis such as rheumatoid arthritis [29]. In addition to the ability to induce RANKL, the inflammatory cytokine synergizes with RANKL in a unique manner. RANKL and TNF each activate a number of key downstream effector pathways, leading to nuclear localization of a range of osteoclastogenic transcription factors [10].
T‐cell cytokines including interferon‐γ (IFN‐γ), IL‐4, and IL‐10 are potent suppressors of osteoclast formation [18,30].
These findings seem to be in conflict with other in vivo observations in which activated T‐cell immune responses are associated with enhanced bone resorption. This is partly because T cells are divided into several subsets with dis- tinct cytokine production. The detailed studies revealed that Th1 cells (producing IFN‐γ) and Th2 cells (producing IL‐4) inhibit osteoclast formation, but Th17 cells stimulate osteoclastogenesis through IL‐17‐mediated induction of RANKL as well as induction of other inflammatory cytokines [31]. IFN‐γ treatment of children with osteopetro- sis ameliorates the disease [32], but IFN‐γ was used to res- cue the immunodeficiency associated with osteopetrosis not to increase the osteoclast number. It is necessary to interpret the in vivo data by taking into consideration vari- ous conditions in addition to the in vitro effect of each cytokine to osteoclastogenesis.
Many additional studies have implicated a range of other soluble factors and cytokines in the regulation of the osteoclast. These include a range of interleukins,
Factors Regulating steoclast Formation anddor Function 51 granulocyte macrophage colony‐stimulating factor
(GM‐CSF), IFN‐β, stromal cell‐derived factor 1 (SDF‐1), macrophage inflammatory protein-1α (MIP-1α), mono- cyte chemoattractant protein 1 (MCP‐l), transforming growth factor β (TGF‐β), various Toll‐like receptor ligands, Wnt ligands, and semaphorins [18,33,34].
Small molecules
1,25‐dihydroxyvitamin D has all the characteristics of a steroid hormone, including a high‐affinity nuclear receptor that binds as a heterodimer with the retinoid X receptor to regulate transcription of a set of specific target genes. This active form of vitamin D, generated by successive hydroxylation in the liver and kidney, is a well‐established stimulator of bone resorption when pre- sent at supraphysiological levels. Studies over many years have indicated that this steroid hormone increases mesenchymal cell transcription of the RANKL gene, while diminishing that of OPG [35]. Separately, 1,25‐
dihydroxyvitamin D suppresses synthesis of the pro‐oste- oclastogenic hormone PTH and enhances calcium uptake from the gut [35]. Taken together, the two latter effects would seem to be antiresorptive, but many studies in humans indicate the net osteolytic action resulting from high levels of this steroid hormone, suggesting that its ability to stimulate osteoclast function overrides any bone anabolic actions.
Loss of estrogen (E2), most often observed in the con- text of menopause, is a major reason for the development of significant bone loss in aging. Interestingly, it is now clear that estrogen is the main sex steroid regulating bone mass in both men and women [36]. The mechanisms by which estrogen mediates its osteolytic effects are still incompletely understood, but significant advances have been made over the last decade. The original hypothesis, now considered to be only part of the explanation, is that decreased serum E2 led to increased production, by circu- lating macrophages, of osteoclastogenic cytokines such as IL‐6, TNF, and IL‐1. These molecules act on stromal cells and osteoclast precursors to enhance bone resorp- tion by regulating expression of pro‐ (RANKL, M‐CSF) and anti‐ (OPG) osteoclastogenic cytokines (in the case of mesenchymal cells) and by synergizing with RANKL itself (in the case of myeloid osteoclast precursors).
However, recent studies suggest other targets of E2 such as T cells and osteoclast lineage cells [36–38]. Further studies are needed to evaluate the relative contribution of E2 effects on these multiple cell types.
Both endogenous glucocorticoids and their synthetic analogs, which have been and continue to be a major mainstay of immunosuppressive therapy, are members of a third steroid hormone family having a major impact on bone biology [39]. One consequence of their chronic mode of administration is severe osteoporosis arising from decreased bone formation and resorption with the latter absolutely decreased (low turnover osteoporosis).
The majority of evidence focuses on the osteoblast as the
prime target with the steroid increasing apoptosis of these bone‐forming cells. However, numerous human studies document a rapid initial decrease in bone resorp- tion, suggesting that the osteoclast and/or its precursors may also be targets. The molecular basis for this latter finding is unclear. However, because osteoblasts are a requisite part of the resorptive cycle, one consequence of their long‐term diminution could be decreased osteoclast formation and/or function secondary to lower levels of RANKL and/or M‐CSF production. Alternatively, glucocorticoids have been shown to decrease osteoclast apoptosis [40].
A wide range of clinical information shows that excess prostaglandins stimulate bone loss, but once again, the cellular basis has not been established. Prostaglandins tar- get stromal and osteoblastic cells, stimulating expression of RANKL and suppressing that of OPG [41]. This increase in the RANKL/OPG ratio, observed in a variety of human studies, is sufficient of itself to explain the clinical findings of increased osteoclastic activity. However, highlighting again the dilemma of interpreting in vitro studies, there have been a number of studies in which prostaglandins regulate osteoclastogenesis per se in murine cell culture.
Phosphoinositides play distinct and important roles in organization of the osteoclast cytoskeleton [42]. Binding of M‐CSF or RANKL to their cognate receptors, c‐Fms and RANK, or activation of ανβ3, recruits phosphoinositol‐3‐
kinase (PI3K) to the plasma membrane, where it converts membrane‐bound phosphatidylinositol 4,5‐bisphosphate into phosphatidylinositol 3,4,5‐ trisphosphate (Fig. 6.3).
The latter compound is recognized by specific motifs in a wide range of cytoskeletally active proteins, and thus PI3K plays a central role in organizing the cytoskeleton of the osteoclast, including its ruffled membrane [42]. Akt is a downstream target of PI3K and plays an important role in osteoclast function, particularly by mediating RANKL and/or M‐CSF‐stimulated proliferation and/or survival [42].
Cell–cell interactions in bone marrow
Recent evidence has indicated that a number of additional cell types are important for osteoclast biology in a variety of situations. First, as discussed previously, T cells play a key role in estrogen deficiency bone loss but also are important in a range of inflammatory diseases, most notably rheumatoid arthritis [18] and periodontal disease [43]; here the Th17 subset likely secretes TNF and IL‐17, a newly described osteoclastogenic cytokine [31]. Given that both osteoclast precursors and the various lympho- cyte subsets, such as T, B, and NK cells, arise from the same stem cell, it is not surprising that some of the same receptors and ligands that mediate the immune process also govern the maturation of osteoclast precursors and the capacity of the mature cell to degrade bone. This interface has given rise to the new discipline of osteoim- munology, which promises to provide important and exciting findings in the future [10,18,33,44].
Second, whereas it is well established that mesenchy- mal cells are major mediators of cytokine and pro- staglandin action on osteoclasts, it has become clear recently that cells of the same lineage, residing on corti- cal and trabecular bone, comprise the site of a HSC niche [44–46]. Specifically, HSCs reside close to osteoblasts as a result of multiple interactions involving receptors and ligands on both cells types [47]. Furthermore, the mesen- chymally derived cells secrete both membrane‐bound and soluble factors that contribute to survival and prolif- eration of multipotent osteoclast precursors, as well as molecules that influence osteoclast formation and function. Both committed osteoblasts and the numerous stromal cells in bone marrow produce a range of proteins both basally and in response to hormones and growth factors, resulting in modulation of the capacity of HSCs to become functional osteoclasts.
Third, cancer cells facilitate their infiltration into the marrow cavity by stimulating osteoclast formation and function. An initial stimulus is PTHrP generation by lung and breast cancer cells [27,28,48], thus enhancing mesenchymal production of RANKL and M‐
CSF, whereas decreasing that of OPG and possibly chemotactic factors. The resulting increase in matrix dissolution releases bone‐residing cytokines and growth factors that, feeding back on the cancer cells, increase their growth and/or survival. This loop has been termed
“the vicious cycle” [27]. Multiple myeloma seems to use a different but related strategy, namely secretion of MIP-1α and MCP‐1, both of which are chemotactic and proliferative for osteoclast precursor [49,50]. The latter compound has been reported to be secreted by osteo- clasts in response to RANKL and enhances osteoclast formation [8]. It seems likely that future studies will uncover additional molecules mediating bone loss in metastatic disease.
CONCLUSION
Osteoclasts act in and elicit changes in the complex bone microenvironment. These cells have a crucial role in maintaining bone health, and they participate in essen- tial reciprocal interactions with osteoblasts, osteocytes, and immune cells to maintain homeostasis. Although many of the key factors that regulate osteoclast forma- tion and activity have been identified, there are many remaining unknowns. We still do not understand how the primary modulators of osteoclastogenesis function or dysfunction in healthy bone and in disease states. The mechanisms underlying different responses of osteo- clasts in different bones or in different bone compart- ments are not well understood. The role of aging in the accumulation of epigenetic changes that affect the activ- ity of osteoclasts has not been elucidated. Answers to these and other questions will expand therapeutic options for the treatment of bone loss in osteoporosis and meta- bolic bone disease.
ACKNOWLEDGMENT
The author is indebted to Dr F. Patrick Ross, who wrote the previous version of this chapter, upon which this update is based.
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Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism, Ninth Edition. Edited by John P. Bilezikian.
© 2019 American Society for Bone and Mineral Research. Published 2019 by John Wiley & Sons, Inc.
Companion website: www.wiley.com/go/asbmrprimer
INTRODUCTION
Mesenchymal stem cells (MSCs) are pluripotent cells located in the bone marrow, muscles, and fat that potentially can differentiate into all mesenchymal tissues. Differentiation towards these cell lineages is controlled by a multitude of cytokines, which regulate the expression of cell‐lineage‐specific sets of transcrip- tion factors. Osteoblasts and chondrocytes are thought to differentiate from a common mesenchymal precursor, the osteochondrogenic precursor. The osteoblastic differ- entiation process can be divided into several stages, including proliferation, extracellular matrix deposition, matrix maturation, and mineralization (Fig. 7.1).