Notum Deletion From Late-Stage Skeletal Cells Increases Cortical Bone Formation and Potentiates Skeletal Effects of Sclerostin Inhibition.

Wnt signaling plays a vital role in the cell biology of skeletal patterning, differentiation, and maintenance. Notum is a secreted member of the α/ß-hydrolase superfamily that hydrolyzes the palmitoleoylate modification on Wnt proteins, thereby disrupting Wnt signaling. As a secreted inhibitor of Wnt, Notum presents an attractive molecular target for improving skeletal health. To determine the cell type of action for Notum's effect on the skeleton, we generated mice with Notum deficiency globally (Notum-/- ) and selectively (Notumf/f ) in limb bud mesenchyme (Prx1-Cre) and late osteoblasts/osteocytes (Dmp1-Cre). Late-stage deletion induced increased cortical bone properties, similar to global mutants. Notum expression was enhanced in response to sclerostin inhibition, so dual inhibition (Notum/sclerostin) was also investigated using a combined genetic and pharmacologic approach. Co-suppression increased cortical properties beyond either factor alone. Notum suppressed Wnt signaling in cell reporter assays, but surprisingly also enhanced Shh signaling independent of effects on Wnt. Notum is an osteocyte-active suppressor of cortical bone formation that is likely involved in multiple signaling pathways important for bone homeostasis © 2021 American Society for Bone and Mineral Research (ASBMR).

The mTORC2 Component Rictor Is Required for Load-Induced Bone Formation in Late-Stage Skeletal Cells.

Bone relies on mechanical cues to build and maintain tissue composition and architecture. Our understanding of bone cell mechanotransduction continues to evolve, with a few key signaling pathways emerging as vital. Wnt/ß-catenin, for example, is essential for proper anabolic response to mechanical stimulation. One key complex that regulates ß-catenin activity is the mammalian target of rapamycin complex 2 (mTORc2). mTORc2 is critical for actin cytoskeletal reorganization, an indispensable component in mechanotransduction in certain cell types. In this study, we probed the impact of the mTORc2 signaling pathway in osteocyte mechanotransduction by conditionally deleting the mTORc2 subunit Rictor in Dmp1-expressing cells of C57BL/6 mice. Conditional deletion of the Rictor was achieved using the Dmp1-Cre driver to recombine Rictor floxed alleles. Rictor mutants exhibited a decrease in skeletal properties, as measured by DXA, µCT, and mechanical testing, compared with Cre-negative floxed littermate controls. in vivo axial tibia loading conducted in adult mice revealed a deficiency in the osteogenic response to loading among Rictor mutants. Histological measurements of osteocyte morphology indicated fewer, shorter cell processes in Rictor mutants, which might explain the compromised response to mechanical stimulation. In summary, inhibition of the mTORc2 pathway in late osteoblasts/osteocytes leads to decreased bone mass and mechanically induced bone formation. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Twist1 Inactivation in Dmp1-Expressing Cells Increases Bone Mass but Does Not Affect the Anabolic Response to Sclerostin Neutralization.

Wnt signaling plays a major role in bone metabolism. Advances in our understanding of secreted regulators of Wnt have yielded several therapeutic targets to stimulate osteoanabolism-the most promising of which is the Wnt inhibitor sclerostin. Sclerostin antibody recently gained approval for clinical use to treat osteoporosis, but the biology surrounding sclerostin antagonism is still incompletely understood. Numerous factors regulate the efficacy of sclerostin inhibition on bone formation, a process known as self-regulation. In previous communications we reported that the basic helix-loop-helix transcription factor Twist1-a gene know to regulate skeletal development-is highly upregulated among the osteocyte cell population in mice treated with sclerostin antibody. In this communication, we tested the hypothesis that preventing Twist1 upregulation by deletion of Twist1 from late-stage osteoblasts and osteocytes would increase the efficacy of sclerostin antibody treatment, since Twist1 is known to restrain osteoblast activity in many models. Twist1-floxed loss-of-function mice were crossed to the Dmp1-Cre driver to delete Twist1 in Dmp1-expressing cells. Conditional Twist1 deletion was associated with a mild but significant increase in bone mass, as assessed by dual energy x-ray absorptiometry (DXA) and microCT (µCT) for many endpoints in both male and female mice. Biomechanical properties of the femur were not affected by conditional mutation of Twist1. Sclerostin antibody improved all bone properties significantly, regardless of Twist1 status, sex, or endpoint examined. No interactions were detected when Twist1 status and antibody treatment were examined together, suggesting that Twist1 upregulation in the osteocyte population is not an endogenous mechanism that restrains the osteoanabolic effect of sclerostin antibody treatment. In summary, Twist1 inhibition in the late-stage osteoblast/osteocyte increases bone mass but does not affect the anabolic response to sclerostin neutralization.