There is currently a demand for new highly efficient and specific drugs to treat osteoporosis, a chronic bone disease affecting millions of people worldwide. We have developed a combinatorial strategy for engineering bispecific inhibitors that simultaneously target the unique combination of c-FMS and α_vβ₃ integrin, which act in concert to facilitate bone resorption by osteoclasts. Using functional fluorescence-activated cell sorting (FACS)-based screening assays of random mutagenesis macrophage colony-stimulating factor (M-CSF) libraries against c-FMS and α_vβ₃ integrin, we engineered dual-specific M-CSF mutants with high affinity to both receptors. These bispecific mutants act as functional antagonists of c-FMS and α_vβ₃ integrin activation and hence of osteoclast differentiation in vitro and osteoclast activity in vivo. This study thus introduces a versatile platform for the creation of new-generation therapeutics with high efficacy and specificity for osteoporosis and other bone diseases. It also provides new tools for studying molecular mechanisms and the cell signaling pathways that mediate osteoclast differentiation and function. Many bone diseases—including osteoporosis, in which the bones become brittle and fragile from loss of tissue—are characterized by excessive and uncontrolled bone resorption by bone-destroying cells known as osteoclasts. Therefore, controlled and specific inhibition of osteoclast activity is a desired outcome in treatments for bone diseases. Osteoclast differentiation and function are coordinated by cell surface receptors, including c-FMS and α_vβ₃ integrin, which cooperate with one another to drive signals that are essential for osteoclast functions. Here, we describe the engineering, characterization, and testing of novel proteins that can target and inhibit both c-FMS and α_vβ₃ integrin at the same time, thereby providing a way of controlling osteoclast function. The study represents the first example of engineering a natural ligand, which acts as a signaling molecule, as a scaffold for binding not only its target protein but also a second target. We show that these engineered proteins inhibit osteoclast activity in a mouse model of osteoporosis. Our study describes potential inhibitors that target all the known functions resulting from c-FMS/integrin α_vβ₃ crosstalk and paves the way to create novel targeting proteins that could be used to treat osteoporosis. It also expands our understanding of the role of the c-FMS/α_vβ₃ integrin pathway in the regulation of osteoclast differentiation and function.

Changes in bone matrix composition are frequently found with bone diseases and maybe associated with increased fracture risk. Bone is rich in the trace element zinc. Zinc was established to play a significant role in the growth, development, and maintenance of healthy bones; however, the mechanisms underlying zinc effects on the integrity of the skeleton are poorly understood. Here, we show that the zinc receptor (ZnR)/Gpr39 is required for normal bone matrix deposition by osteoblasts. Initial analysis showed that Gpr39‐deficient ( Gpr39 ^−/− ) mice had weaker bones as a result of altered bone composition. Fourier transform infrared spectroscopy analysis showed high mineral‐to‐matrix ratios in the bones of Gpr39 ^−/− mice. Histologic analysis showed abnormally high numbers of active osteoblasts but normal osteoclast numbers on the surfaces of bones from Gpr39 ^−/− mice. Furthermore, Gpr39 ^−/− osteoblasts had disorganized matrix deposition in vitro with cultures exhibiting abnormally low collagen and high mineral contents, findings that demonstrate a cell‐intrinsic role for ZnR/Gpr39 in these cells. We show that both collagen synthesis and deposition by Gpr39 ^−/− osteoblasts are perturbed. Finally, the expression of the zinc transporter Zip13 and a disintegrin and metalloproteinase with thrombospondin motifs family of zinc‐dependent metalloproteases that regulate collagen processing was downregulated in Gpr39 ^−/− osteoblasts. Altogether, our results suggest that zinc sensing by ZnR/Gpr39 affects the expression levels of zinc‐dependent enzymes in osteoblasts and regulates collagen processing and deposition.—Jovanovic, M., Schmidt, F. N., Guterman‐Ram, G., Khayyeri, H., Hiram‐Bab, S., Orenbuch, A., Katchkovsky, S., Aflalo, A., Isaksson, H, Busse, B., Jahn, K., Levaot, N. Perturbed bone composition and integrity with disorganized osteoblast function in zinc receptor/Gpr39‐deficient mice. FASEB J. 32, 2507–2518 (2018). www.fasebj.org

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. Slfn2^eka/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 Slfn2^eka/eka mice showed a reduction in osteoclast number and total resorption area. Finally, we show that the bone marrow of Slfn2^eka/eka mice have significantly less CD11b^–Ly6C^hi 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.