Notch signaling between neighboring cells controls many cell fate decisions in metazoans both during embryogenesis and in postnatal life. Previously, we uncovered a critical role for physiological Notch signaling in suppressing osteoblast differentiation in vivo. However, the contribution of individual Notch receptors and the downstream signaling mechanism have not been elucidated. Here we report that removal of Notch2, but not Notch1, from the embryonic limb mesenchyme markedly increased trabecular bone mass in adolescent mice. Deletion of the transcription factor RBPjk, a mediator of all canonical Notch signaling, in the mesenchymal progenitors but not the more mature osteoblast-lineage cells, caused a dramatic high-bone-mass phenotype characterized by increased osteoblast numbers, diminished bone marrow mesenchymal progenitor pool, and rapid age-dependent bone loss. Moreover, mice deficient in Hey1 and HeyL, two target genes of Notch-RBPjk signaling, exhibited high bone mass. Interestingly, Hey1 bound to and suppressed the NFATc1 promoter, and RBPjk deletion increased NFATc1 expression in bone. Finally, pharmacological inhibition of NFAT alleviated the high-bone-mass phenotype caused by RBPjk deletion. Thus, Notch-RBPjk signaling functions in part through Hey1-mediated inhibition of NFATc1 to suppress osteoblastogenesis, contributing to bone homeostasis in vivo.

The bone morphogenetic protein (Bmp) family of secreted molecules has been extensively studied in the context of osteoblast differentiation. However, the intracellular signaling cascades that mediate the osteoblastogenic function of Bmp have not been fully elucidated. By profiling mRNA expression in the bone marrow mesenchymal progenitor cell line ST2, we discover that BMP2 induces not only genes commonly associated with ossification and mineralization but also genes important for general protein synthesis. We define the two groups of genes as mineralization related versus protein anabolism signatures of osteoblasts. Although it induces the expression of several Wnt genes, BMP2 activates the osteogenic program largely independently of de novo Wnt secretion. Remarkably, although Smad4 is necessary for the activation of the mineralization-related genes, it is dispensable for BMP2 to induce the protein anabolism signature, which instead critically depends on the transcription factor Atf4. Upstream of Atf4, BMP2 activates mTORC1 to stimulate protein synthesis, resulting in an endoplasmic reticulum stress response mediated by Perk. Thus, Bmp signaling induces osteoblast differentiation through both Smad4-and mTORC1-dependent mechanisms.

Exogenous bone morphogenetic proteins (Bmp) are well known to induce ectopic bone formation, but the physiological effect of Bmp signaling on normal bone is not completely understood. By deleting the receptor Bmpr1a in osteoblast lineage cells with Dmp1-Cre, we observed a dramatic increase in trabecular bone mass in postnatal mice, which was due to a marked increase in osteoblast number that was likely to be driven by hyperproliferation of Sp7 + preosteoblasts. Similarly, inducible deletion of Bmpr1a in Sp7 + cells specifically in postnatal mice increased trabecular bone mass. However, deletion of Smad4 by the same approaches had only a minor effect, indicating that Bmpr1a signaling suppresses trabecular bone formation through effectors beyond Smad4. Besides increasing osteoblast number in the trabecular bone, deletion of Bmpr1a by Dmp1-Cre also notably reduced osteoblast activity, resulting in attenuation of periosteal bone growth. The impairment in osteoblast activity correlated with reduced mTORC1 signaling in vivo, whereas inhibition of mTORC1 activity abolished the induction of protein anabolism genes by BMP2 treatment in vitro. Thus, physiological Bmpr1a signaling in bone exerts a dual function in both restricting preosteoblast proliferation and promoting osteoblast activity.

High fracture rate and high circulating levels of the Wnt inhibitor, sclerostin, have been reported in diabetic patients. We studied the effects of Wnt signaling activation on bone health in a mouse model of insulin-deficient diabetes. We introduced the sclerostinresistant Lrp5 A214V mutation, associated with high bone mass, in mice carrying the Ins2 Akita mutation (Akita), which results in loss of beta cells, insulin deficiency, and diabetes in males. Akita mice accrue less trabecular bone mass with age relative to wild type (WT). Double heterozygous Lrp5 A214V /Akita mutants have high trabecular bone mass and cortical thickness relative to WT animals, as do Lrp5 A214V single mutants. Likewise, the Lrp5 A214V mutation prevents deterioration of biomechanical properties occurring in Akita mice. Notably, Lrp5 A214V /Akita mice develop fasting hyperglycemia and glucose intolerance with a delay relative to Akita mice (7 to 8 vs. 5 to 6 weeks, respectively), despite lack of insulin production in both groups by 6 weeks of age. Although insulin sensitivity is partially preserved in double heterozygous Lrp5 A214V /Akita relative to Akita mutants up to 30 weeks of age, insulin-dependent phosphorylated protein kinase B (pAKT) activation in vitro is not altered by the Lrp5 A214V mutation. Although white adipose tissue depots are equally reduced in both compound and Akita mice, the Lrp5 A214V mutation prevents brown adipose tissue whitening that occurs in Akita mice. Thus, hyperactivation of Lrp5-dependent signaling fully protects bone mass and strength in prolonged hyperglycemia and improves peripheral glucose metabolism in an insulin independent manner. Wnt signaling activation represents an ideal therapeutic approach for diabetic patients at high risk of fracture.