Osteoclasts are multinucleated bone resorbing cells formed by cytoplasmic fusion of their mononuclear precursors, which are in the myeloid lineage of hematopoietic cells that also give rise to macrophages. The switch to osteoclast differentiation requires expression in osteoclast precursors (OCPs) of c-Fos, a RANKL activated transcription factor [3
]. To resorb bone effectively, osteoclasts attach themselves firmly to the bone surface using specialized actin-rich podosomes, which they use to form tightly sealed roughly circular extensions of their cytoplasm with the underlying bone matrix. Within these sealed zones they form ruffled membranes that increase the surface area of the cell membrane for secretion of hydrochloric acid and the proteolytic enzyme cathepsin K onto the bone surface [4
]. They thereby simultaneously dissolve the mineral and degrade the matrix of bone, while protecting neighboring cells from harm by this sealing mechanism. They are activated by RANKL and by integrin-mediated signaling from bone matrix itself [4
]. Osteoclasts work in packs within remodeling units under the control of osteoblast lineage cells expressing macrophage colony-stimulating factor (M-CSF) and RANKL.
Recent studies of the mechanisms by which PTH exerts its anabolic effects have suggested that osteoclasts are probably involved in the recruitment of packs of bone-forming osteoblasts to refill the trenches that they form on the bone surface [2
]. This is based on studies showing that, after PTH injection, RANKL expression is increased by osteoblast/stromal cells, leading to activation of existing osteoclasts and release by them of a factor(s) that stimulates new bone formation. Also, antiresorptive treatment, at least in some studies, appears to reduce rather than enhance the anabolic action of PTH [5
]. As is discussed below, osteoclasts also appear to regulate immune responses and their own production at sites of inflammation in bone, such as rheumatoid joints.
Osteoclasts are required during embryonic development for the removal of bone trabeculae formed under growth plates during endochondral ossification and thus for formation of the bone marrow cavity to facilitate normal hematopoiesis. Failure of osteoclast formation or activity results in osteopetrosis, some forms of which are lethal because of attendant immunodeficiency and increased risk for infection and recurrent fractures. Indeed, the development of osteopetrosis in a variety of knockout mice has identified necessary functions of genes in osteoclast biology that largely had not been anticipated [3
Our understanding of the molecular mechanisms that regulate osteoclast formation and activation has advanced rapidly during the past 10 years since the discovery of the RANKL/RANK signaling system, and following the development in the late 1980s of in vitro
assays that facilitated harvesting of large numbers of OCPs from bone marrow or spleen cells, which could then be cultured in the absence of osteoblast/stromal cells. The strategy for acquiring OCPs from these sources was developed in the knowledge that M-CSF expression by osteoblast/stromal cells was required for progenitor cells to differentiate into osteoclasts, but that M-CSF on its own was unable to complete this process. This requirement for M-CSF was based on the observation that op/op mice, which do not express functional M-CSF, have osteopetrosis because of a lack of osteoclasts [3
]. Indeed, since 1981, when Rodan and Martin [1
] proposed the novel hypothesis that osteoblast/stromal cells play a central role in the regulation of osteoclast formation and bone resorption, many investigators had attempted to identify the osteoclast-activating factor that completed the differentiation of precursors that had been exposed to M-CSF.
Discovery of osteoprotegerin, RANKL, and RANK
Between 1981 and the mid 1990s, the Rodan–Martin hypothesis was supported by many studies, but the factor(s) expressed by osteoblast/stromal or other cells remained undetermined until they were discovered independently by four groups using different approaches.
Boyle and coworkers [8
] at Amgen Inc. (Thousand Oaks, CA, USA) discovered OPG unexpectedly in studies to identify tumor necrosis factor (TNF) receptor related molecules with possible therapeutic utility by generating transgenic mice that over-express various TNF receptor related cDNAs. Mice over-expressing one particular cDNA developed marked osteopetrosis because they did not have any osteoclasts in their bones. The protein encoded by the gene was named osteoprotegerin (the bone protector) [8
], because it appeared to protect the skeleton from excessive bone resorption by limiting osteoclastic bone resorption. Independently, researchers at the Snow Brand Milk Products Co. (Sapporo, Hokkaido, Japan) reported their discovery of an identical molecule [9
] using the standard approach to test the Rodan–Martin hypothesis of purifying a factor from human embryonic fibroblasts that inhibited osteoclastogenesis. They obtained a partial protein sequence and subsequently cloned the cDNA for OPG.
Using expression cloning and OPG as a probe, both groups quickly identified its ligand, which they called OPG ligand and osteoclast differentiation factor, respectively [10
]. This ligand turned out to be identical to a member of the TNF ligand family, which had been identified in the preceding year as RANKL [12
] and TNF-related activation induced cytokine [13
]. Soon after OPG ligand/osteoclast differentiation factor was identified as a ligand for OPG, the cellular receptor was identified as being identical to the previously identified RANK, which Anderson and coworkers [12
] at Immunex (Seattle, WA, USA) had discovered while they were sequencing cDNAs from a human bone marrow derived myeloid dendritic cell cDNA library. They found that RANK had partial homology to a portion of the extracellular domain of human CD40, a member of the TNF receptor superfamily, and that it was involved in the activation of T cells in the immune system. They then isolated RANKL by direct expression screening and found, like Wong and coworkers [13
] did, that it increased dendritic cell stimulated naïve T cell proliferation and survival of RANK-expressing T cells. These discoveries that RANKL is involved in osteoclastogenesis and T cell activation have spawned the now growing field of osteoimmunology.