The skeleton is a dynamic tissue continuously adapting to mechanical stimuli. Although matrix‐embedded osteocytes are considered as the key mechanoresponsive bone cells, all other skeletal cell types are principally exposed to macroenvironmental and microenvironmental mechanical influences that could potentially affect their activities. It was recently reported that Piezo1, one of the two mechanically activated ion channels of the Piezo family, functions as a mechanosensor in osteoblasts and osteocytes. Here we show that Piezo1 additionally plays a critical role in the process of endochondral bone formation. More specifically, by targeted deletion of Piezo1 or Piezo2 in either osteoblast (Runx2Cre) or osteoclast lineage cells (Lyz2Cre), we observed severe osteoporosis with numerous spontaneous fractures specifically in Piezo1^Runx2Cre mice. This phenotype developed at an early postnatal stage and primarily affected the formation of the secondary spongiosa. The presumptive Piezo1^Runx2Cre osteoblasts in this region displayed an unusual flattened appearance and were positive for type X collagen. Moreover, transcriptome analyses of primary osteoblasts identified an unexpected induction of chondrocyte‐related genes in Piezo1^Runx2Cre cultures. Because Runx2 is not only expressed in osteoblast progenitor cells, but also in prehypertrophic chondrocytes, these data suggested that Piezo1 functions in growth plate chondrocytes to ensure trabecular bone formation in the process of endochondral ossification. To confirm this hypothesis, we generated mice with Piezo1 deletion in chondrocytes (Col2a1Cre). These mice essentially recapitulated the phenotype of Piezo1^Runx2Cre animals, because they displayed early‐onset osteoporosis with multiple fractures, as well as impaired formation of the secondary spongiosa with abnormal osteoblast morphology. Our data identify a previously unrecognized key function of Piezo1 in endochondral ossification, which, together with its role in bone remodeling, suggests that Piezo1 represents an attractive target for the treatment of skeletal disorders. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Bone growth requires a specialised, highly angiogenic blood vessel subtype, so-called type H vessels, which pave the way for osteoblasts surrounding these vessels. At the end of adolescence, type H vessels differentiate into quiescent type L endothelium lacking the capacity to promote bone growth. Until now, the signals that switch off type H vessel identity and thus limit adolescent bone growth have remained ill defined. Here we show that mechanical forces, associated with increased body weight at the end of adolescence, trigger the mechanoreceptor PIEZO1 and thereby mediate enhanced production of the kinase FAM20C in osteoblasts. FAM20C, the major kinase of the secreted phosphoproteome, phosphorylates dentin matrix protein 1, previously identified as a key factor in bone mineralization. Thereupon, dentin matrix protein 1 is secreted from osteoblasts in a burst-like manner. Extracellular dentin matrix protein 1 inhibits vascular endothelial growth factor signalling by preventing phosphorylation of vascular endothelial growth factor receptor 2. Hence, secreted dentin matrix protein 1 transforms type H vessels into type L to limit bone growth activity and enhance bone mineralization. The discovered mechanism may suggest new options for the treatment of diseases characterised by aberrant activity of bone and vessels such as osteoarthritis, osteoporosis and osteosarcoma.

Skeletal growth, modeling, and remodeling are regulated by various molecules, one of them being the recently identified osteoanabolic factor WNT1. We have previously reported that WNT1 transcriptionally activates the expression of Omd, encoding Osteomodulin (OMD), in a murine mesenchymal cell line, which potentially explained the skeletal fragility of mice with mutational WNT1 inactivation, since OMD has been shown to regulate type I collagen fibril formation in vitro. In this study we confirmed the strong induction of Omd expression in a genome-wide expression analysis of transfected cells, and we obtained further evidence for Omd being a direct target gene of WNT1. To assess the in vivo relevance of this regulation, we crossed Omd-deficient mice with a mouse line harboring an inducible, osteoblast-specific Wnt1 transgene. After induction of Wnt1 expression for 1 or 3 weeks, the osteoanabolic potency of WNT1 was not impaired despite the Omd deficiency. Since current knowledge regarding the in vivo physiological function of OMD is limited, we next focused on skeletal phenotyping of wild-type and Omd-deficient littermates, in the absence of a Wnt1 transgene. Here we did not observe an impact of Omd deficiency on trabecular bone parameters by histomorphometry and μCT either. Importantly, however, male and female Omd-deficient mice at the ages of 12 and 24 weeks displayed a slender bone phenotype with significantly smaller long bones in the transversal dimension, while the longitudinal bone growth remained unaffected. Although mechanical testing revealed no significant changes explained by impaired bone material properties, atomic force microscopy of the femoral bone surface of Omd-deficient mice revealed moderate changes at the nanostructural level, indicating altered regulation of collagen fibril formation and aggregation. Taken together, our data demonstrate that, although OMD is dispensable for the osteoanabolic effect of WNT1, its deficiency in mice specifically modulates transversal cortical bone morphology. We explored the physiological relevance of the protein Osteomodulin (OMD) that we previously found to be induced by the osteoanabolic molecule WNT1. While other studies have shown that OMD is involved in the regulation of collagen fibril formation in vitro, its function in vivo has not been investigated. We confirmed that OMD is directly regulated by WNT1 but surprisingly, when we bred mice lacking OMD with mice engineered to highly express WNT1, we found that the osteoanabolic effect of WNT1 was unaffected by the absence of OMD. Interestingly, mice lacking OMD did show differences in the shape of their bones, particularly in their width, despite no significant changes in bone density or length. Investigation of the bone matrix of mice lacking OMD at the nanostructural level indicated moderate differences in the organization of collagen fibrils. This study provided further insights into the effect of WNT1 on bone metabolism and highlighted a specific function of OMD in skeletal morphology.

Breast cancer cells frequently metastasize to bone, where their interaction with bone remodeling cell types enhances osteolytic bone destruction. Importantly, however, whereas skeletal analyses of xenograft models are usually restricted to hindlimb bones, human skeletal metastases are far more frequent in the spine, where trabecular bone mass is higher compared to femur or tibia. Here, we addressed whether breast cancer cells injected into immunocompromised mice metastasize to the spine and if this process is influenced by the amount of trabecular bone. We also took advantage of mice carrying the Col1a1-Krm2 transgene, which display severe osteoporosis. After crossing this transgene into the immunocompromised NSG background we injected MDA-MB-231-SCP2 breast cancer cells and analyzed their distribution three weeks thereafter. We identified more tumor cells and clusters of different size in spine sections than in femora, which allowed influences on bone remodeling cell types to be analyzed by comparing tumor-free to tumor-burdened areas. Unexpectedly, the Col1a1-Krm2 transgene did not affect spreading and metastatic outgrowth of MDA-MB-231-SCP2 cells, suggesting that bone tumor interactions are more relevant at later stages of metastatic progression.

Albeit osteoporosis is one of the most prevalent disorders in the aged population, treatment options stimulating the activity of bone-forming osteoblasts are still limited. We and others have previously identified sphingosine-1-phosphate (S1P) as a bone remodeling coupling factor, which is released by bone-resorbing osteoclasts to stimulate bone formation. Moreover, S1pr3, encoding one of the five known S1P receptors (S1P3), was found differentially expressed in osteoblasts, and S1P3 deficiency corrected the moderate high bone mass phenotype of a mouse model (deficient for the calcitonin receptor) with increased S1P release from osteoclasts. In the present study we addressed the question, if S1P3 deficiency would also influence the skeletal phenotype of mice lacking S1P-lyase (encoded by Sgpl1), which display markedly increased S1P levels due to insufficient degradation. Consistent with previous reports, the majority of Sgpl1-deficient mice died before or shortly after weaning, and this lethality was not influenced by additional S1P3 deficiency. At 3 weeks of age, Sgpl1-deficient mice displayed increased trabecular bone mass, which was associated with enhanced osteoclastogenesis and bone resorption, but also with increased bone formation. Most importantly however, none of the skeletal parameters assessed by μCT, histomorphometry and serum analyses were significantly influenced by additional S1P3 deficiency. Taken together, our findings fully support the concept that S1P is a potent osteoanabolic molecule, although S1P3 is not the sole receptor mediating this influence. Since S1P receptors are considered excellent drug targets, it is now required to screen for the impact of other family members on bone formation.

Bone growth requires a specialised, highly angiogenic blood vessel subtype, so-called type H vessels^1,2, which pave the way for osteoblasts surrounding these vessels³. At the end of adolescence, type H endothelial cells differentiate into quiescent type L endothelium lacking the capacity to promote bone growth. Until now, the signals that switch off type H vessel identity and thus arrest adolescent bone growth have remained ill defined. Here we show that mechanical forces, associated with increased body weight at the end of adolescence, trigger the mechanoreceptor PIEZO1 and thereby mediate enhanced production of the kinase FAM20C in osteoblasts. FAM20C phosphorylates dentin matrix protein 1 (DMP1)⁴, previously identified as a key factor in bone mineralization⁵. This phosphorylation elicits a burst in DMP1 secretion from osteoblasts. Extracellular DMP1 inhibits vascular endothelial growth factor (VEGF) signalling by preventing VEGFR2 phosphorylation and VEGFR3 expression on the tip cells of type H endothelium. DMP1-mediated VEGF inhibition transforms bone growth-promoting type H vessels into quiescent type L vasculature to arrest bone growth and enhance bone mineralization. This molecular mechanism links mechanical forces and the termination of bone growth via accumulation of an extracellular matrix protein and its regulation of vascular subtypes. It suggests new options for the treatment of diseases characterised by inappropriate turnover or invasion of bone such as osteoarthritis, osteoporosis and osteosarcoma.