Bone resorption goes green

Abstract

In this issue of Cell, McDonald et al. show that giant multinucleated, bone-resorbing osteoclasts dissolve into smaller cells, termed “osteopmorhs,” which re-form into osteoclasts at distal bone sites (McDonald et al., 2021). These findings overturn the long-standing premise that osteoclasts differentiate solely from hematopoietic precursors and undergo apoptosis after completing resorption.

The osteoclast is unique in its ability to remove old bone. This resorptive function matches precisely the requirements of skeletal morphogenesis, modeling, and remodeling. The latter, a process described by the orthopedic surgeon John Hunter as early as 1743, is defined by the constant removal of old bone and its replenishment by an equal amount of new bone by the osteoblast (Hunter, 1743). Enhanced osteoclastic resorption, reduced osteoblastic bone formation, or a combination of both results in a net loss of bone, as in osteoporosis or skeletal metastasis—and consequently, a high risk of fracture (Zaidi, 2007).

Much of our current understanding of osteoclast biology originates from studies in the 1970s demonstrating through parabiosis experiments that osteoclasts are derived from hematopoietic cells (Buring, 1975). Subsequently, two key cytokines were discovered to be necessary and sufficient for osteoclast formation from hematopoietic cells: macrophage colonystimulating factor (M-CSF) and receptor activator for nuclear factor kB ligand (RANKL) (Lacey et al., 1998). These studies underscore the concept that precursors of a monocytic lineage fuse together to form terminally differentiated multinuclear polykaryons that no longer have the ability to proliferate but can resorb bone (Zaidi et al., 2018). The lifespan of osteoclasts is thought to be relatively short, around 2 to 3 weeks, with their demise resulting from apoptosis following the cessation of resorption at a given site. However, contemporary work in rodents has called into question this view of a fleeting lifespan for osteoclasts. In parabiosis experiments using genetically modified mice that expressed two different fluorescent markers, it was shown recently that hematopoietic cells from one animal not only became incorporated into the osteoclasts of the other animal, as had been shown in the 1970’s, but that their nuclei continued to be present in osteoclasts for up to 6 months after the animals were separated (Jacome-Galarza et al., 2019).

These findings challenged the premise that osteoclasts undergo apoptosis after resorption and supported an alternative notion that they were instead longlived cells. If osteoclasts are enduring and not transitory in nature, what happens to osteoclasts that are no longer needed at a resorption site? Similarly, how are new ones formed at distal sites needing resorption? To help address these questions, McDonald et al. (2021) developed a novel technique using fluorescent reporters to image osteoclast formation and fate in the tibias of adult mice (McDonald et al., 2021). Specifically, they repleted the bone marrows of irradiated mice with a combination of cells from two different donor mice––one that expressed tdTomato in LYSM+ myeloid cells and the other from mice engineered to express GFP driven by either the Csf1r or Blimp-1 promoters. The fusion of these two cells to form a multinucleate osteoclast allowed them to be tracked with two fluorescent signals.

Using this technique, the authors were able to penetrate the cortex of mouse tibias to find that osteoclasts were stellate in appearance and underwent dynamic fission events into smaller, motile daughter cells, which they termed ‘‘osteomorphs.’’ These daughter osteomorphs were then observed to migrate and fuse into nearby osteoclasts in a relatively balanced process, where around 1% of osteoclasts per hour experienced either a fusion or fission event. The phenomenon of osteoclast fission is not new, having been first described in the 1980s (Baron and Vignery, 1981). However, the fate of these daughter cells following fission has remained largely unstudied.

To determine whether osteoporphs acquired a distinct transcriptomic architecture from osteoclasts, McDonald et al. (2021) performed single-cell RNA sequencing on flow cytometry-sorted fluorescent mononuclear osteomorphs that had previously resorbed bone in vivo, as evidenced by the uptake during resorption of a third fluorescent marker, the bisphosphonate zolendronic acid. They found that these cells had a unique transcriptional program, with marrow osteomorphs expressing many of the traditional osteoclast genes, such as Ctsk (cathepsin K), Acp5 (tartrateresistant acid phosphatase, TRAP), and Nfatc1 (NFAT2), with an overall transcriptomic concordance of 75% between osteomorphs and osteoclasts. Nonetheless, 151 genes were expressed only in osteomorphs and not in osteoclasts. Loss-of-function mutations in certain of these osteomorph-specific genes, such as bisphosphoglycerate mutase (Bpgm) and F-box protein 7 (Fbxo 7), are known to produce bone phenotypes in mice. These findings together suggest that certain genes can be targeted specifically in osteomorphs to yield functional, and possibly therapeutic, outcomes.

Notwithstanding transcriptomic distinctions, the molecular regulation of osteoclast fission into osteomorphs has not yet been examined. There does not appear to be an easy way to extract osteomorphs from the marrow, which will complicate future efforts to study these cells. McDonald et al. (2021) note that osteomorphs displayed a higher expression than monocytic precursors of the cell-surface markers AXL, CD11b, CCR3, VCAM1, CD74, and CADM1; however, it remains to be seen if these markers can be used to separate osteomorphs from marrow monocytic cells. One interesting avenue for potential exploration arises from the fact that osteomorphs appear to circulate in the bloodstream. Potential targeting of these cells in the control of arterial plaques, which are known to have osteoclast-like cells (Qiao et al., 2015), may unravel new avenues for the treatment of arterial disease.

The authors next began to ask whether this process of osteoclast conservation bears relevance to current osteoporosis therapeutics. They show that osteomorphs accumulate within bone marrow upon treatment with OPG:Fc, an agent that blocks RANKL and closely mimics the actions of the FDA-approved osteoporosis therapeutic denosumab. Upon withdrawal of the RANKL inhibition, however, these cells rapidly fused to form boneresorbing osteoclasts—a finding that helps explain the sharp increases in fracture risk following discontinuation of denosumab therapy in people with osteoporosis (Anastasilakis et al., 2020). This further reiterates the suggestion that cessation of denosumab treatment should be followed by an osteoclast apoptosis-inducing agent, such as a bisphosphonate, to prevent osteomorphs from rapidly fusing into osteoclasts with rebound bone loss.

In summary, the study by McDonald et al. (2021) uncovers a novel cellular program for the reuse of osteoclasts, notably through their dissolution, transport, and reassembly (Figure 1). We expect this discovery to usher in a new wave of greater insights into osteoclast physiology, as well as to unmask new actionable targets during different phases of the osteoclast life cycle.

Figure 1. Cellular recycling in bone with giant osteoclasts broken down into smaller osteomorphs allowing for their reuse at other resorption sites.

Large multinucleated osteoclasts are the only cells known to degrade bone in a process termed resorption. It has been surmised that when resorption is no longer required at a particular bone site, osteoclasts undergo apoptosis. Nonetheless, McDonald et al. (2021) show that the normal process is for osteoclasts to undergo a fission event into smaller cells called osteomorphs. These smaller daughter cells are transcriptionally distinct from osteoclasts and can either remain in waiting to be called when needed, or alternatively travel to other sites in bone where resorption is required, where they fuse together to recreate a giant multinucleated osteoclast