Summary Cherubism is an autosomal dominant syndrome characterized by inflammatory destructive bony lesions resulting in symmetrical deformities of the facial bones. Cherubism is caused by mutations in Sh3bp2, the gene that encodes the adaptor protein 3BP2. Most identified mutations in 3BP2 lie within the peptide sequence RSPPDG. A mouse model of cherubism develops hyperactive bone remodelling osteoclasts and systemic inflammation characterized by expansion of the myelomonocytic lineage. The mechanism by which cherubism mutations alter 3BP2 function has remained obscure. Here we show that Tankyrase, a member of the poly(ADP-ribose)polymerase (PARP) family, regulates 3BP2 stability through ADP-ribosylation and subsequent ubiquitylation by the E3-ubiquitin ligase RNF146 in osteoclasts. Cherubism mutations uncouple 3BP2 from Tankyrase-mediated protein destruction, which results in its stabilization and subsequent hyperactivation of the SRC, SYK and VAV signalling pathways.

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 cellintrinsic 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. Introduction3BP2 is an adapter protein that contains an N-terminal pleckstrin homology (PH) domain, a proline-rich stretch that binds to Src homology 3 (SH3) domain-containing proteins, and a C-terminal SH2 domain that binds to phosphotyrosine residues (1). 3BP2 was initially identified as a binding protein of the tyrosine kinase Abl (2). Work from our lab and others has identified the Src family kinases (SFKs), Syk, and the Vav family of Rho guanine nucleotide exchange factors (GEFs) as 3BP2-binding partners (1), all of which are known to play important roles in osteoclast function (3-5). Cherubism is a dominantly inherited syndrome characterized by excessive maxillary and mandibular bone resorption that is associated with activated osteoclasts and inflammatory cells creating interosseous cystic lesions (6). Single missense mutations in the gene encoding the adapter protein 3BP2 result in a gain-of-function alteration in the protein and is associated with the majority of cherubism patients (7). A mouse model that harbors 2 copies of a cherubism allele develops severe osteoporosis associated with highly activated osteoclasts (8).In order to elucidate the role of the wild-type form of 3BP2 in bone homeostasis, we analyzed loss-of-function mutant mice. As distinct from the osteoporotic phenotype of mice expressing the cherubism gain-of-function form of 3BP2 associated with active osteoclasts, we uncovered a complex bone phenotype in mice lacking 3BP2 characterized by loss-of-function in both the osteoclast and osteoblast lineages resulting in net decreased bone mineral density and reduced mechanical ...

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The transcription factor TWIST1 plays a vital role in mesoderm development, particularly in limb and craniofacial formation. Accordingly, haploinsufficiency of TWIST1 can cause limb and craniofacial malformations as part of Saethre-Chotzen syndrome. However, the molecular basis of TWIST1 transcriptional regulation during development has yet to be elucidated. Here, we characterized active enhancers in the TWIST1-HDAC9 locus that drive transcription in the developing limb and branchial arches. Using available p300 and H3K27ac ChIP-seq data, we identified 12 enhancer candidates, located both within and outside the coding sequences of the neighboring gene, Histone deacetyase 9 (HDAC9). Using zebrafish and mouse enhancer assays, we showed that eight of these candidates have limb/fin and branchial arch enhancer activity that resemble Twist1 expression. Using 4C-seq, we showed that the Twist1 promoter region interacts with three enhancers (eTw-5, 6, 7) in the limb bud and branchial arch of mouse embryos at day 11.5. Furthermore, we found that two transcription factors, LMX1B and TFAP2, bind these enhancers and modulate their enhancer activity. Finally, using CRISPR/Cas9 genome editing, we showed that homozygous deletion of eTw5-7 enhancers reduced Twist1 expression in the limb bud and caused pre-axial polydactyly, a phenotype observed in Twist1+/- mice. Taken together, our findings reveal that each enhancer has a discrete activity pattern, and together comprise a spatiotemporal regulatory network of Twist1 transcription in the developing limbs/fins and branchial arches. Our study suggests that mutations in TWIST1 enhancers could lead to reduced TWIST1 expression, resulting in phenotypic outcome as seen with TWIST1 coding mutations.

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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 αvβ3 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 αvβ3 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 αvβ3 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.

Changes in bone matrix composition are frequently found with bone diseases and may be 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., Jähn, K., Levaot, N. Perturbed bone composition and integrity with disorganized osteoblast function in zinc receptor/Gpr39-deficient mice.