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Osteocalcin (OCN), the most abundant noncollagenous protein in the bone matrix, is reported to be a bone-derived endocrine hormone with wide-ranging effects on many aspects of physiology, including glucose metabolism and male fertility. Many of these observations were made using an OCN-deficient mouse allele (Osc-) in which the 2 OCN-encoding genes in mice, Bglap and Bglap2, were deleted in ES cells by homologous recombination. Here we describe mice with a new Bglap and Bglap2 double-knockout (dko) allele (Bglap/2p.Pro25fs17Ter) that was generated by CRISPR/Cas9-mediated gene editing. Mice homozygous for this new allele do not express full-length Bglap or Bglap2 mRNA and have no immunodetectable OCN in their serum. FTIR imaging of cortical bone in these homozygous knockout animals finds alterations in the collagen maturity and carbonate to phosphate ratio in the cortical bone, compared with wild-type littermates. However, μCT and 3-point bending tests do not find differences from wild-type littermates with respect to bone mass and strength. In contrast to the previously reported OCN-deficient mice with the Osc-allele, serum glucose levels and male fertility in the OCN-deficient mice with the Bglap/2pPro25fs17Ter allele did not have significant differences from wild-type littermates. We cannot explain the absence of endocrine effects in mice with this new knockout allele. Possible explanations include the effects of each mutated allele on the transcription of neighboring genes, or differences in genetic background and environment. So that our findings can be confirmed and extended by other interested investigators, we are donating this new Bglap and Bglap2 double-knockout strain to the Jackson Laboratories for academic distribution.

Mutations in the Wnt co-receptor, LRP5 , lead to skeletal diseases in humans. Matthew Warman and his colleagues have now developed mutant mice with tissue-specific alterations of Lrp5 expression and found that these mice phenocopy the human skeletal diseases. They also found that Lrp5 acts locally to affect bone homeostasis. Their data suggest that increasing LRP5 signaling in mature bone cells may be a strategy to treat osteoporosis. The human skeleton is affected by mutations in low-density lipoprotein receptor-related protein 5 (LRP5). To understand how LRP5 influences bone properties, we generated mice with osteocyte-specific expression of inducible Lrp5 mutations that cause high and low bone mass phenotypes in humans. We found that bone properties in these mice were comparable to bone properties in mice with inherited mutations. We also induced an Lrp5 mutation in cells that form the appendicular skeleton but not in cells that form the axial skeleton; we observed that bone properties were altered in the limb but not in the spine. These data indicate that Lrp5 signaling functions locally, and they suggest that increasing LRP5 signaling in mature bone cells may be a strategy for treating human disorders associated with low bone mass, such as osteoporosis.

Autosomal dominant hypophosphatemic rickets (ADHR) is unique among the disorders involving Fibroblast growth factor 23 (FGF23) because individuals with R176Q/W and R179Q/W mutations in the FGF23 176RXXR179/S180 proteolytic cleavage motif can cycle from unaffected status to delayed onset of disease. This onset may occur in physiological states associated with iron deficiency, including puberty and pregnancy. To test the role of iron status in development of the ADHR phenotype, WT and R176Q-Fgf23 knock-in (ADHR) mice were placed on control or low-iron diets. Both the WT and ADHR mice receiving low-iron diet had significantly elevated bone Fgf23 mRNA. WT mice on a low-iron diet maintained normal serum intact Fgf23 and phosphate metabolism, with elevated serum C-terminal Fgf23 fragments. In contrast, the ADHR mice on the low-iron diet had elevated intact and C-terminal Fgf23 with hypophosphatemic osteomalacia. We used in vitro iron chelation to isolate the effects of iron deficiency on Fgf23 expression. We found that iron chelation in vitro resulted in a significant increase in Fgf23 mRNA that was dependent upon Mapk. Thus, unlike other syndromes of elevated FGF23, our findings support the concept that late-onset ADHR is the product of gene–environment interactions whereby the combined presence of an Fgf23-stabilizing mutation and iron deficiency can lead to ADHR.

Wnt signaling pathways are transmitted via 10 homologous frizzled receptors (FZD1-10) in humans. Reagents broadly inhibiting Wnt signaling pathways reduce growth and metastasis of many tumors, but their therapeutic development has been hampered by the side effect. Inhibitors targeting specific Wnt-FZD pair(s) enriched in cancer cells may reduce side effect, but the therapeutic effect of narrow-spectrum Wnt-FZD inhibitors remains to be established in vivo. Here, we developed a fragment of C . difficile toxin B (TcdB FBD ), which recognizes and inhibits a subclass of FZDs, FZD1/2/7, and examined whether targeting this FZD subgroup may offer therapeutic benefits for treating breast cancer models in mice. Utilizing 2 basal-like and 1 luminal-like breast cancer models, we found that TcdB FBD reduces tumor-initiating cells and attenuates growth of basal-like mammary tumor organoids and xenografted tumors, without damaging Wnt-sensitive tissues such as bones in vivo. Furthermore, FZD1/2/7–positive cells are enriched in chemotherapy-resistant cells in both basal-like and luminal mammary tumors treated with cisplatin, and TcdB FBD synergizes strongly with cisplatin in inhibiting both tumor types. These data demonstrate the therapeutic value of narrow-spectrum Wnt signaling inhibitor in treating breast cancers.

Osteocytes, former osteoblasts buried within bone, are thought to orchestrate skeletal adaptation to mechanical stimuli. However, it remains unknown whether hormones control skeletal homeostasis through actions on osteocytes. Parathyroid hormone (PTH) stimulates bone remodeling and may cause bone loss or bone gain depending on the balance between bone resorption and formation. Herein, we demonstrate that transgenic mice expressing a constitutively active PTH receptor exclusively in osteocytes exhibit increased bone mass and bone remodeling, as well as reduced expression of the osteocyte-derived Wnt antagonist sclerostin, increased Wnt signaling, increased osteoclast and osteoblast number, and decreased osteoblast apoptosis. Deletion of the Wnt co-receptor LDL related receptor 5 (LRP5) attenuates the high bone mass phenotype but not the increase in bone remodeling induced by the transgene. These findings demonstrate that PTH receptor signaling in osteocytes increases bone mass and the rate of bone remodeling through LRP5-dependent and -independent mechanisms, respectively.

The low density lipoprotein receptor-related protein-5 (LRP5), a co-receptor in the Wnt signaling pathway, modulates bone mass in humans and in mice. Lrp5 knock-out mice have severely impaired responsiveness to mechanical stimulation whereas Lrp5 gain-of-function knock-in and transgenic mice have enhanced responsiveness to mechanical stimulation. Those observations highlight the importance of Lrp5 protein in bone cell mechanotransduction. It is unclear if and how high bone mass-causing (HBM) point mutations in Lrp5 alter the bone-wasting effects of mechanical disuse. To address this issue we explored the skeletal effects of mechanical disuse using two models, tail suspension and Botulinum toxin-induced muscle paralysis, in two different Lrp5 HBM knock-in mouse models. A separate experiment employing estrogen withdrawal-induced bone loss by ovariectomy was also conducted as a control. Both disuse stimuli induced significant bone loss in WT mice, but Lrp5 A214V and G171V were partially or fully protected from the bone loss that normally results from disuse. Trabecular bone parameters among HBM mice were significantly affected by disuse in both models, but these data are consistent with DEXA data showing a failure to continue growing in HBM mice, rather than a loss of pre-existing bone. Ovariectomy in Lrp5 HBM mice resulted in similar protection from catabolism as was observed for the disuse experiments. In conclusion, the Lrp5 HBM alleles offer significant protection from the resorptive effects of disuse and from estrogen withdrawal, and consequently, present a potential mechanism to mimic with pharmaceutical intervention to protect against various bone-wasting stimuli.

The L‐enantiomer of β‐aminoisobutyric acid (BAIBA) is secreted by contracted muscle in mice, and exercise increases serum levels in humans. In mice, L‐BAIBA reduces bone loss with unloading, but whether it can have a positive effect with loading is unknown. Since synergism can be more easily observed with sub‐optimal amounts of factors/stimulation, we sought to determine whether L‐BAIBA could potentiate the effects of sub‐optimal loading to enhance bone formation. L‐BAIBA was provided in drinking water to C57Bl/6 male mice subjected to either 7 N or 8.25 N of sub‐optimal unilateral tibial loading for 2 weeks. The combination of 8.25 N and L‐BAIBA significantly increased the periosteal mineral apposition rate and bone formation rate compared to loading alone or BAIBA alone. Though L‐BAIBA alone had no effect on bone formation, grip strength was increased, suggesting a positive effect on muscle function. Gene expression analysis of the osteocyte‐enriched bone showed that the combination of L‐BAIBA and 8.25 N induced the expression of loading‐responsive genes such as Wnt1, Wnt10b, and the TGFb and BMP signaling pathways. One dramatic change was the downregulation of histone genes in response to sub‐optimal loading and/or L‐BAIBA. To determine early gene expression, the osteocyte fraction was harvested within 24 hours of loading. A dramatic effect was observed with L‐BAIBA and 8.25 N loading as genes were enriched for pathways regulating the extracellular matrix (Chad, Acan, Col9a2), ion channel activity (Scn4b, Scn7a, Cacna1i), and lipid metabolism (Plin1, Plin4, Cidec). Few changes in gene expression were observed with sub‐optimal loading or L‐BAIBA alone after 24 hours. These results suggest that these signaling pathways are responsible for the synergistic effects between L‐BAIBA and sub‐optimal loading. Showing that a small muscle factor can enhance the effects of sub‐optimal loading of bone may be of relevance for individuals unable to benefit from optimal exercise. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research. The muscle metabolite L‐BAIBA synergizes with sub‐optimal mechanical loading of the mouse tibia to promote new bone formation and regulate the transcriptional response of osteocyte‐enriched cortical bone.

Administration of sclerostin-neutralizing antibody (Scl-Ab) treatment has been shown to elicit an anabolic bone response in growing and adult mice. Prior work characterized the response of individual mouse strains but did not establish whether the impact of Scl-Ab on whole bone strength would vary across different inbred mouse strains. Herein, we tested the hypothesis that two inbred mouse strains (A/J and C57BL/6J (B6)) will show different whole bone strength outcomes following sclerostin-neutralizing antibody (Scl-Ab) treatment during growth (4.5-8.5 weeks of age). Treated B6 femurs showed a significantly greater stiffness (S) (68.8% vs. 46.0%) and maximum load (ML) (84.7% vs. 44.8%) compared to A/J. Although treated A/J and B6 femurs showed greater cortical area (Ct.Ar) similarly relative to their controls (37.7% in A/J and 41.1% in B6), the location of new bone deposition responsible for the greater mass differed between strains and may explain the greater whole bone strength observed in treated B6 mice. A/J femurs showed periosteal expansion and endocortical infilling, while B6 femurs showed periosteal expansion. Post-yield displacement (PYD) was smaller in treated A/J femurs (-61.2%, p < 0.001) resulting in greater brittleness compared to controls; an effect not present in B6 mice. Inter-strain differences in S, ML, and PYD led to divergent changes in work-to-fracture (Work). Work was 27.2% (p = 0.366) lower in treated A/J mice and 66.2% (p < 0.001) greater in treated B6 mice relative to controls. Our data confirmed the anabolic response to Scl-Ab shown by others, and provided evidence suggesting the mechanical benefits of Scl-Ab administration may be modulated by genetic background, with intrinsic growth patterns of these mice guiding the location of new bone deposition. Whether these differential outcomes will persist in adult and elderly mice remains to be determined.

Advanced prostate cancer frequently metastasizes to bone, but no effective therapy exists. To seek a novel treatment option and identify a new drug target, we took an induced tumor-suppressing (iTS) cell-based approach and produced tumor-suppressing proteins and conditioned medium (CM). Notably, the overexpression of Lrp5 and β-catenin, as well as the pharmacological Wnt activator, converted osteocytes, Murine mesenchymal stem cells, mononuclear cells, and monocytes into iTS cells. While Lrp5 conditional knockout mice presented severe bone loss, Lrp5-overexpressing osteocyte-derived CM rescued tumor-induced bone damage. Whole-genome proteomics analysis revealed that Moesin (MSN), which acted as an oncogene in tumor cells, was enriched in CM as an extracellular tumor-suppressing protein. Its anti-tumor action was mediated primarily by the interaction with CD44. Consistently, FRET live-cell imaging demonstrated that extracellular MSN reduced Src tyrosine kinase activity and nuclear localization of β-catenin. Collectively, we demonstrated herein the iTS cell-based approach to protect bone from prostate cancer and showed MSN as a potent extracellular tumor-suppressing protein.