Osteosarcoma (OS) is the most frequent primary malignant bone tumor in urgent need of better therapies. Using genetically modified mouse models (GEMMs), we demonstrate that Wnt signaling promotes c-Fos-induced OS formation via the actions of the collagen-modifying enzyme Loxl2. c-Fos/AP-1 directly regulates the expression of the Wnt ligands Wnt7b and Wnt9a in OS cells through promoter binding, and Wnt7b and Wnt9a in turn promote Loxl2 expression in murine and human OS cells through the transcription factors Zeb1 and Zeb2. Concordantly, inhibition of Wnt ligand secretion by inactivating the Wnt-less (Wls) gene in osteoblasts in c-Fos GEMMs either early or in a therapeutic setting reduces Loxl2 expression and progression of OS. Wls-deficient osteosarcomas proliferate less, are less mineralized and are enriched in fibroblastic cells surrounded by collagen fibers. Importantly, Loxl2 inhibition using either the pan-Lox inhibitor BAPN or a specific inducible shRNA reduces OS cell proliferation in vitro and decreases tumor growth and lung colonization in murine and human orthotopic OS transplantation models. Finally, OS development is delayed in c-Fos GEMMs treated with BAPN or with specific Loxl2 blocking antibodies. Congruently, a strong correlation between c-FOS, LOXL2 and WNT7B/WNT9A expression is observed in human OS samples, and c-FOS/LOXL2 co-expression correlates with OS aggressiveness and decreased patient survival. Therefore, therapeutic targeting of Wnt and/or Loxl2 should be considered to potentiate the inadequate current treatments for pediatric, recurrent, and metastatic OS.

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Mutations in the human receptor tyrosine kinase ROR2 are associated with Robinow syndrome (RRS) and brachydactyly type B1. Amongst others, the shortened limb phenotype associated with RRS is recapitulated in Ror2−/− mutant mice. In contrast, Ror1−/− mutant mice are viable and show no limb phenotype. Ror1−/−;Ror2−/− double mutants are embryonic lethal, whereas double mutants containing a hypomorphic Ror1 allele (Ror1hyp) survive up to birth and display a more severe shortened limb phenotype. Both orphan receptors have been shown to act as possible Wnt coreceptors and to mediate the Wnt5a signal. Here, we analyzed genetic interactions between the Wnt ligand, Wnt9a, and Ror2 or Ror1, as Wnt9a has also been implicated in skeletal development. Wnt9a−/− single mutants display a mild shortening of the long bones, whereas these are severely shortened in Ror2−/− mutants. Ror2−/−;Wnt9a−/− double mutants displayed even more severely shortened long bones, and intermediate phenotypes were observed in compound Ror2;Wnt9a mutants. Long bones were also shorter in Ror1hyp/hyp;Wnt9a−/− double mutants. In addition, Ror1hyp/hyp;Wnt9a−/− double mutants displayed a secondary palate cleft phenotype, which was not present in the respective single mutants. Interestingly, 50% of compound mutant pups heterozygous for Ror2 and homozygous mutant for Ror1 also developed a secondary palate cleft phenotype.

The formation of the bone marrow cavity is a prerequisite for endochondral ossification. In reviews and textbooks, it is occasionally reported that osteoclasts are essential for bone marrow cavity formation removing hypertrophic chondrocytes. Mice lacking osteoclasts or having functionally defective osteoclasts have osteopetrotic bones, yet they still form a bone marrow cavity. Here, we investigated the role of osteoclasts and macrophages in bone marrow cavity formation during embryogenesis. Macrophages can assist osteoclasts in matrix removal by phagocytosing resorption byproducts. Rank-deficient mice, lacking osteoclasts, and Pu.1-deficient mice, lacking monocytes, macrophages, and osteoclasts, displayed a delay in bone marrow cavity formation and a lengthening of the zone of hypertrophic chondrocytes. F4/80-positive monocyte/macrophage numbers increased by about fourfold in the bone marrow cavity of E18.5 Rank-deficient mice. Based on lineage-tracing experiments, the majority of the excess F4/80 cells were derived from definitive hematopoietic precursors of the fetal liver. In long bones of both Rank À/À and Pu.1 À/À specimens, Mmp9-positive cells were still present. In addition to monocytes, macrophages, and osteoclasts, Ctsb-positive septoclasts were lost in Pu.1 À/À specimens. The mineralization pattern was altered in Rank À/À and Pu.1 À/À specimens, revealing a significant rise in transverse-oriented mineralized structures. Taken together, our findings imply that early on during bone marrow cavity formation, osteoclasts facilitate the entry of blood vessels and later the turnover of hypertrophic chondrocytes, whereas macrophages appear to play no major role. Furthermore, the absence of septoclasts in Pu.1 À/À specimens suggests that septoclasts are either derived from Pu.1-dependent precursors or require PU.1 activity for their differentiation.

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