Noam Levaot

3BP2-deficient mice are osteoporotic with impaired osteoblast and osteoclast functions

A fine balance between bone resorption by osteoclasts and bone formation by osteoblasts maintains bone homeostasis. In patients with cherubism, gain-of-function mutations in 3BP2, which is encoded by SH3-domain binding protein 2 (SH3BP2), cause cystic lesions with activated osteoclasts that lead to craniofacial abnormalities. However, little is known about the function of wild-type 3BP2 in regulating bone homeostasis. Here we have shown that 3BP2 is required for the normal function of both osteoblasts and osteoclasts. Initial analysis showed that Sh3bp2-/-mice developed osteoporosis as a result of reduced bone formation despite the fact that bone resorption was impaired. We demonstrated using reciprocal bone marrow chimeras, a cell-intrinsic defect of the osteoblast and osteoclast compartments in vivo. Further, Sh3bp2-/- osteoblasts failed to mature and form mineralized nodules in vitro, while Sh3bp2-/- osteoclasts spread poorly and were unable to effectively degrade dentine matrix in vitro. Finally, we showed that 3BP2 was required for Abl activation in osteoblasts and Src activation in osteoclasts, and demonstrated that the in vitro defect of each cell type was restored by the respective expression of activated forms of these kinases. These findings reveal an unanticipated role for the 3BP2 adapter protein in osteoblast function and in coordinating bone homeostatic signals in both osteoclast and osteoblast lineages.

Combinatorial and computational approaches to identify interactions of macrophage colony stimulating factor (M-CSF) and its receptor c-fms

Background: Identifying residues crucial for M-CSF·c-FMS binding remains difficult. Results: Using a combination of experimental and computational methods, we identified mutations on M-CSF that reduce affinity to c-FMS. Conclusion: Affinity-reducing mutations are located both inside and outside of the binding interface. Significance: Knowledge of the critical residues will facilitate a better understanding of the M-CSF mechanism and facilitate drug design. The molecular interactions between macrophage colony-stimulating factor (M-CSF) and the tyrosine kinase receptor c-FMS play a key role in the immune response, bone metabolism, and the development of some cancers. Because no x-ray structure is available for the human M-CSF·c-FMS complex, the binding epitope for this complex is largely unknown. Our goal was to identify the residues that are essential for binding of the human M-CSF to c-FMS. For this purpose, we used a yeast surface display (YSD) approach. We expressed a combinatorial library of monomeric M-CSF (M-CSFM) single mutants and screened this library to isolate variants with reduced affinity for c-FMS using FACS. Sequencing yielded a number of single M-CSFM variants with mutations both in the direct binding interface and distant from the binding site. In addition, we used computational modeling to map the identified mutations onto the M-CSFM structure and to classify the mutations into three groups as follows: those that significantly decrease protein stability; those that destroy favorable intermolecular interactions; and those that decrease affinity through allosteric effects. To validate the YSD and computational data, M-CSFM and three variants were produced as soluble proteins; their affinity and structure were analyzed; and very good correlations with both YSD data and computational predictions were obtained. By identifying the M-CSFM residues critical for M-CSF·c-FMS interactions, we have laid down the basis for a deeper understanding of the M-CSF·c-FMS signaling mechanism and for the development of target-specific therapeutic agents with the ability to sterically occlude the M-CSF·c-FMS binding interface.

Engineering a monomeric variant of macrophage colony-stimulating factor (M‑CSF) that antagonizes the c-FMS receptor

Enhanced activation of the signaling pathways that mediate the differentiation of mononuclear monocytes into osteoclasts is an underlying cause of several bone diseases and bone metastasis. In particular, dysregulation and overexpression of macrophage colony-stimulating factor (M-CSF) and its c-FMS tyrosine kinase receptor, proteins that are essential for osteoclast differentiation, are known to promote bone metastasis and osteoporosis, making both the ligand and its receptor attractive targets for therapeutic intervention. With this aim in mind, our starting point was the previously held concept that the potential of the M-CSFC31S mutant as a therapeutic is derived from its inability to dimerize and hence to act as an agonist. The current study showed, however, that dimerization is not abolished in M-CSFC31S and that the protein retains agonistic activity toward osteoclasts. To design an M-CSF mutant with diminished dimerization capabilities, we solved the crystal structure of the M-CSFC31S dimer complex and used structure-based energy calculations to identify the residues responsible for its dimeric form. We then used that analysis to develop M-CSFC31S,M27R, a ligand-based, high-affinity antagonist for c-FMS that retained its binding ability but prevented the ligand dimerization that leads to receptor dimerization and activation. The monomeric properties of M-CSFC31S,M27R were validated using dynamic light scattering and small-angle X-ray scattering analyses. It was shown that this mutant is a functional inhibitor of M-CSF-dependent c-FMS activation and osteoclast differentiation in vitro Our study, therefore, provided insights into the sequence-structure-function relationships of the M-CSF/c-FMS interaction and of ligand/receptor tyrosine kinase interactions in general.

Dual‐specificity tyrosine phosphorylation‐regulated kinase 2 regulates osteoclast fusion in a cell heterotypic manner

Monocyte fusion into osteoclasts, bone resorbing cells, plays a key role in bone remodeling and homeostasis; therefore, aberrant cell fusion may be involved in a variety of debilitating bone diseases. Research in the last decade has led to the discovery of genes that regulate osteoclast fusion, but the basic molecular and cellular regulatory mechanisms underlying the fusion process are not completely understood. Here, we reveal a role for Dyrk2 in osteoclast fusion. We demonstrate that Dyrk2 down regulation promotes osteoclast fusion, whereas its overexpression inhibits fusion. Moreover, Dyrk2 also promotes the fusion of foreign‐body giant cells, indicating that Dyrk2 plays a more general role in cell fusion. In an earlier study, we showed that fusion is a cell heterotypic process initiated by fusion‐founder cells that fuse to fusion‐follower cells, the latter of which are unable to initiate fusion. Here, we show that Dyrk2 limits the expansion of multinucleated founder cells through the suppression of the fusion competency of follower cells.

Schlafen2 mutation in mice causes an osteopetrotic phenotype due to a decrease in the number of osteoclast progenitors

Osteoclasts are the bone resorbing cells that derive from myeloid progenitor cells. Although there have been recent advancements in the ability to identify osteoclast progenitors, very little is known about the molecular mechanisms governing their homeostasis. Here, by analyzing the normalized phylogenetic profiles of the Schlafen (Slfn) gene family, we found that it co-evolved with osteoclast-related genes. Following these findings, we used a Slfn2 loss-of-function mutant mouse, elektra, to study the direct role of Slfn2 in osteoclast development and function. Slfn2eka/eka mice exhibited a profound increase in their cancellous bone mass and a significant reduction in osteoclast numbers. In addition, monocyte cultures from the bone marrow of Slfn2eka/eka mice showed a reduction in osteoclast number and total resorption area. Finally, we show that the bone marrow of Slfn2eka/eka mice have significantly less CD11b–Ly6Chi osteoclast precursors. Overall, our data suggest that Slfn2 is required for normal osteoclast differentiation and that loss of its function in mice results in an osteopetrotic phenotype.

How cellular Zn2+ signaling drives physiological functions

Zinc is an essential micronutrient affecting many aspects of human health. Cellular Zn2+ homeostasis is critical for cell function and survival. Zn2+, acting as a first or second messenger, triggers signaling pathways that mediate the physiological roles of Zn2+. Transient changes in Zn2+ concentrations within the cell or in the extracellular region occur following its release from Zn2+ binding metallothioneins, its transport across membranes by the ZnT or ZIP transporters, or release of vesicular Zn2+. These transients activate a distinct Zn2+ sensing receptor, ZnR/GPR39, or modulate numerous proteins and signaling pathways. Importantly, Zn2+ signaling regulates cellular physiological functions such as: proliferation, differentiation, ion transport and secretion. Indeed, novel therapeutic approaches aimed to maintain Zn2+ homeostasis and signaling are evolving. This review focuses on recent findings describing roles of Zn2+ and its transporters in regulating physiological or pathological processes.