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Cell Fusion Stages in Osteoclastogenesis: Mechanisms and Physiological Role

• Leonid V. Chernomordik, PhD, Head, Section on Membrane Biology
• Eugenia Leikina, DVM, Senior Research Assistant
• Kamram Melikov, PhD, Staff Scientist
• Elena Zaitseva, PhD, Staff Scientist
• Jarred Whitlock, PhD, Intramural Research Training Award Fellow
• Griffin Katz, BS, Postbaccalaureate Fellow
• Amit Puthan, BS, Postbaccalaureate Fellow
• Morgan Roegner, BS, Postbaccalaureate Fellow

Diverse biological processes, in which enveloped viruses infect cells, and cells from all kingdoms of life secrete, internalize, traffic and sort integral proteins, sculpt their membranes and bring together parent genomes in sexual reproduction, share a common stage: fusion of two membranes into one. Biological membrane remodeling is tightly controlled by protein machinery, but is also dependent on the lipid composition of the membranes. Whereas each kind of protein has its own individual personality, membrane lipid bilayers have rather general properties, manifested by their resistance to disruption and bending, and by their charge. Our long-term goal is to understand how proteins fuse membrane lipid bilayers. We expect better understanding of important fusion reactions to bring about new ways of controlling them and lead to new strategies for quelling diseases involving cell invasion by enveloped viruses and defects in intracellular trafficking or intercellular fusion. Our general strategy is to combine in-depth analysis of the best characterized fusion reactions with comparative analysis of diverse less explored fusion reactions, which can reveal new kinds of fusion proteins and clarify the generality of emerging mechanistic insights. In our recent studies, we explored the mechanisms of osteoclast fusion in bone remodeling in in vitro models and in our new inducible explant model for dissecting osteoclast fusion and osteoclast-osteoblast coordination in fibrous dysplasia.

Cell surface–bound La protein regulates the cell fusion stage of osteoclastogenesis.

Bone-resorbing osteoclasts are responsible for essential, life-long skeletal remodeling, and their dysfunction is a major contributor to bone diseases, affecting over 200 million individuals worldwide, including osteoporosis, fibrous dysplasia (FD), Paget's disease, and osteopetrosis. Multinucleated osteoclasts are formed by the successive fusion of mononucleated precursor cells, where each fusion event raises the bone-resorbing activity of osteoclasts. Despite the fundamental role of cell-cell fusion in osteoclast formation, the mechanisms of this fusion process in normal physiology and in disease remain to be fully understood. In our recent study [Reference 1], we discovered that osteoclastogenesis involves the lupus La protein (SSB gene product). La, also referred to as LARP3 or La autoantigen, is generally recognized as an abundant and ubiquitous, mostly nuclear RNA–binding protein. The best characterized function of nuclear La is to protect precursor tRNAs from exonuclease digestion through specific interactions between La's highly conserved, N-terminal La domain and the 3′ ends of tRNA.

We found that the La protein has an additional function as an osteoclast fusion regulator. The differentiation of murine and human monocytes into multinucleated osteoclasts is accompanied by and depends on tightly choreographed changes in the steady-state level, post-translational modification, and cellular localization of La. In fusing osteoclasts, La is found at the surface of the cells, and the cell-surface associated La, rather than intracellular La, regulates osteoclast fusion. The changes in the expression of La and the functional role of its cell-surface form in osteoclastogenesis are unexpected in the context of the vast literature covering La’s role in RNA biology. La is generally thought of as an abundant, ubiquitous, mostly phosphorylated RNA–binding protein largely confined to the nucleus in virtually all eukaryotic cell types. However, at the onset of osteoclastogenesis, M-CSF (macrophage colony stimulating factor)–derived precursors show a dramatic loss of La protein. In the following RANKL–induced (RANKL is a protein that controls bone resorption, regeneration, and modeling) stages of osteoclastogenesis, La reappears as a non-phosphorylated, proteolytically cleaved species in the cytoplasm and at the surface of the fusing osteoclast precursors. When the growth of osteoclasts slows, in the late stages of fusion, La is observed at its conventional molecular weight and nuclear localization. The rate of formation, the sizes of multinucleated syncytia, and the subsequent bone resorption activity of osteoclasts are regulated by cell-surface La protein. In fact, cell-surface La regulates osteoclast functions by modulating the membrane fusion stage of osteoclast formation, not upstream differentiation processes. Lowering the amount of La by suppressing the steady-state level of its transcript, blocking its proteolytic processing, or inhibiting its activity with antibodies inhibits fusion. Conversely, increasing La’s steady-state concentration by either overexpression or application of recombinant protein promotes fusion. In addition, the addition of α-La antibodies or recombinant La at the surface of osteoclasts inhibits and promotes synchronized osteoclast fusion, respectively. Importantly, the upregulation of cell-surface La and its involvement in osteoclast fusion have been observed for both primary human and murine osteoclasts, suggesting that La’s role in regulating fusion is conserved in mammals.

The mechanisms by which cell-surface La regulates osteoclast fusion remain to be clarified. Given that La, on its own, initiates neither hemifusion nor fusion between bound membranes, it is unlikely that La directly catalyzes and/or drives membrane fusion. More likely, La recruits or stimulates other components of the osteoclast fusion complex. Our findings highlight La’s association with the fusion regulator phosphatidylserine (PS)–binding protein Annexin A5 (Anx A5). Anx A5 has been implicated in several cell-cell fusion processes. In the case of osteoclast fusion, osteoclastogenic differentiation of human monocytes is associated with a strong increase in the amount of Anx A5 at the cell surface, and treatments suppressing the expression and activity of cell-surface Anx A5 inhibit synchronized osteoclast fusion. We found that recombinant La and Anx A5 directly interact, and that Anx A5 enriches La on membranes containing PS in a Ca²⁺-dependent manner. These observations: (1) explain how La, a soluble protein, associates with cell membranes; (2) connect La function in osteoclastogenesis with the non-apoptotic PS exposure signaling pathway that is thought to trigger osteoclast fusion; (3) and, in combination with the previously reported dependence of osteoclast fusion on cell-surface PS and Anx A5, shed additional light on how osteoclasts employ PS to initiate the assembly of a fusion complex between committed precursors.

In summary, our work demonstrates that La, a key protein in the RNA biology of eukaryotic cells, lives a second life at the surface of osteoclasts, where it moonlights as a master regulator of osteoclast membrane fusion (Figure 1). We suggest that, in this new, highly specific role on the surface of fusing osteoclasts, La may present a promising target for treatment of bone diseases stemming from perturbed bone turnover. Considering that mononucleated osteoclasts do resorb bones, blocking La–dependent osteoclast fusion is expected to have more subtle and selective effects on bone resorption than blocking the upstream formation of osteoclast precursors with α-RANKL antibodies. Cell-surface La is accessible for cell-impermeable drugs. Furthermore, the only known function of cell-surface La is its newly identified role in regulating osteoclast fusion. Surface La’s specificity may minimize off-target effects. The future development of safe and effective reagents targeting cell-surface bound La may lead to novel antiresorptive therapies for osteoporosis, mechanistically orthogonal to the existing approaches.

An inducible explant model of osteoclast-osteoprogenitor coordination in exacerbated osteoclastogenesis

Precise coordination of resorption of the bones by osteoclasts (OCs) and formation of the bones by osteoblasts (OBs) is critically important for maintaining our skeleton throughout our lifespan. Disruption of the coordination of OC and OB functions leads to skeletal diseases. Given that the paucity of simple, activatable, biologically relevant models of osteoclast-osteoblast coordination has hindered our understanding of the osteoblast-osteoclast crosstalk, we developed an inducible ex vivo model of osteoclast-osteoblast progenitor coordination [Reference 2]. Our experimental system utilizes a conditional, tetracycline-inducible mouse model of fibrous dysplasia (FD). Induction of the Gα_s^R201C mutation in osteoprogenitors by treating the cells in bone marrow explants with doxycycline activates the release of osteoclastogenic factors from osteoprogenitors, which, in a RANKL (receptor activator of NF-kB ligand)–dependent manner, elicits the differentiation and fusion of neighboring preosteoclasts. In turn, multinucleated osteoclasts promote osteoprogenitor proliferation by releasing soluble coupling factors and RANKL–positive extracellular vesicles (Figure 2). Our model condenses to days the time course of excessive osteoclast formation typical of FD and many other bone diseases and thus facilitates exploring the mechanism of underlying molecular mechanisms. Although the cellular make-up of the explant cultures described and the biological activities represented are complex, employing this model is relatively simple. The training required to master the required dissections is minimal, we observe high phenotypic reproducibility between cultures and for at least four passages within individual cultures, and the cost in maintaining and implementing this model of osteoclast/osteoprogenitor biology is modest, as the only media additive required for activation is doxycycline. We expect this model to expedite the investigation of cell-cell fusion, osteoclast-osteoblast progenitor coordination, and extracellular vesicle signaling during bone remodeling and to offer a powerful tool for evaluating signaling cascades and novel therapeutic interventions in osteoclast-linked skeletal disease.

Additional Funding

• Office of AIDS Research Award 2023
• Research on Women's Health (ORWH) through the Bench to Bedside Program award #884515. 2022, 2023
• Binational Science Foundation (BSF) Award 2021168 (2023–2026)

Publications

1. Whitlock JM, Leikina E, Melikov K, de Castro, LF, Mattijssen S, Maraia RJ, Collins MT, Chernomordik LV. Cell surface-bound La protein regulates the cell fusion stage of osteoclastogenesis. Nat Commun 2023 14:616.
2. Whitlock JM, de Castro, LF, Collins MT, Chernomordik LV, Boyce AM. An inducible explant model of osteoclast-osteoprogenitor coordination in exacerbated osteoclastogenesis. iScience 2023 26:106470.

Collaborators

• Alison Boyce, MD, Metabolic Bone Disorders Unit, NIDCR, Bethesda, MD
• Michael Collins, MD, Skeletal Disorders & Mineral Homeostasis Section, NIDCR, Bethesda, MD
• Michael M. Kozlov, PhD, DHabil, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
• Richard Maraia, MD, Section on Molecular and Cellular Biology, NICHD, Bethesda, MD
• Leonid Margolis, PhD, Section on Intercellular Interactions, NICHD, Bethesda, MD

Contact

For more information, email chernoml@mail.nih.gov or visit https://www.nichd.nih.gov/research/atNICHD/Investigators/chernomordik.

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