Notch receptors are determinants of cell fate and function and play a central role in skeletal development and bone remodeling. Hajdu Cheney syndrome, a disease characterized by osteoporosis and fractures, is associated with NOTCH2 mutations resulting in a truncated stable protein and gain-of-function. We created a mouse model reproducing the Hajdu Cheney syndrome by introducing a 6955C3 T mutation in the Notch2 locus leading to a Q2319X change at the amino acid level. Notch2Q2319X heterozygous mutants were smaller and had shorter femurs than controls; and at 1 month of age they exhibited cancellous and cortical bone osteopenia. As the mice matured, cancellous bone volume was restored partially in male but not female mice, whereas cortical osteopenia persisted in both sexes. Cancellous bone histomorphometry revealed an increased number of osteoclasts and bone resorption, without a decrease in osteoblast number or bone formation. Osteoblast differentiation and function were not affected in Notch2 Q2319X cells. The pre-osteoclast cell pool, osteoclast differentiation, and bone resorption in response to receptor activator of nuclear factor B ligand in vitro were increased in Notch2 Q2319X mutants. These effects were suppressed by the ␥-secretase inhibitor LY450139. In conclusion, Notch2 Q2319X mice exhibit cancellous and cortical bone osteopenia, enhanced osteoclastogenesis, and increased bone resorption.Notch proteins are four single-pass transmembrane receptors that play a critical role in cell fate decisions ( Fig. 1) (1-4). Notch regulates cell renewal and plays a role in skeletal development and homeostasis and in osteoblast and osteoclast differentiation (4 -8). Jagged1 and -2 and DeltaLike1, -3, and -4 are the five classic Notch ligands (4). Notch-ligand interactions result in the proteolytic cleavage and release of the Notch intracellular domain (NICD), 2 which translocates to the nucleus to form a complex with recombination signal-binding protein for immunoglobulin J region (Rbpj) and Mastermind-like to regulate transcription (9 -12). This canonical signaling pathway leads to the transcription of Hairy Enhancer of Split (Hes)1, -5, and -7 and Hes related with YRPW motif (Hey)1, -2, and -L. Skeletal cells express Notch1, Notch2, and low levels of Notch3 transcripts (13-15). Activation of Notch in undifferentiated and differentiated osteoblasts inhibits cell differentiation and function and causes osteopenia (16,17). In contrast, activation of Notch1 in osteocytes causes a pronounced increase in bone mass due to a suppression of bone resorption (18). Results from the conditional inactivation of Notch1 and Notch2 in the developing skeleton confirmed the inhibitory role of Notch in osteoblastogenesis (6,19). Whereas substantial work has characterized the consequences of Notch1 gain-of-function in the skeleton, there is limited knowledge on the function of Notch2 in the postnatal skeleton. This knowledge is particularly important because Notch1 and Notch2 do not have redundant functions, and Notch1 inhibits, wh...

Lateral meningocele syndrome (LMS) is a rare genetic disorder characterized by neurological complications and osteoporosis. LMS is associated with mutations in exon 33 of NOTCH3 leading to a truncated protein lacking sequences for NOTCH3 degradation and presumably causing NOTCH3 gain-of-function. To create a mouse model reproducing human LMS-associated mutations, we utilized CRISPR/Cas9 to introduce a tandem termination codon at bases 6691-6696 (ACCAAG>TAATGA) and verified this mutation (Notch3 tm1.1Ecan

Individuals with Hajdu-Cheney syndrome (HCS) present with osteoporosis, and HCS is associated with mutations causing deletions of the proline-, glutamic acid-, serine-, and threonine-rich (PEST) domain that are predicted to enhance NOTCH2 stability and cause gain-of-function. Previously, we demonstrated that mice harboring mutations analogous to those in HCS () are severely osteopenic because of enhanced bone resorption. We attributed this phenotype to osteoclastic sensitization to the receptor activator of nuclear factor-κB ligand and increased osteoblastic tumor necrosis factor superfamily member 11 () expression. Here, to determine the individual contributions of osteoclasts and osteoblasts to HCS osteopenia, we created a conditional-by-inversion ( ) model in which Cre recombination generates a allele expressing a Notch2 mutant lacking the PEST domain. Germ line inversion phenocopied the mutant, validating the model. To activate Notch2 in osteoclasts or osteoblasts, mice were bred with mice expressing Cre from the or the promoter, respectively. These crosses created experimental mice harboring a allele in Cre-expressing cells and control littermates expressing a wild-type transcript. inversion in-expressing cells had no skeletal consequences and did not affect the capacity of bone marrow macrophages to form osteoclasts In contrast, inversion in osteoblasts led to generalized osteopenia associated with enhanced bone resorption in the cancellous bone compartment and with suppressed endocortical mineral apposition rate. Accordingly, activation in osteoblast-enriched cultures from mice induced expression. In conclusion, introduction of the HCS mutation in osteoblasts, but not in osteoclasts, causes osteopenia.

We have previously shown that Singleminded-2s (SIM2s), a member of the basic helix-loop-helix Per-Arnt-Sim (bHLH/ PAS) family of transcription factors, is downregulated in breast cancer samples and has tumor suppressor activity. However, the mechanism by which SIM2s is repressed in breast cancer cells has not been determined. In this study, we show that transformation of MCF10A cells by HarveyRas (Ha-Ras) induces CCAAT/enhance binding protein b (C/EBPb) and activates the NOTCH signaling pathway to block SIM2s gene expression. NOTCH-mediated repression acts through a C-repeat binding factor 1 (CBF1)-independent mechanism, as introduction of CBF1 had no effect on SIM2s expression. Consistent with C/ebpbdependent inhibition of SIM2s, C/ebpb À/À mouse mammary glands express high levels of SIM2s and reestablishment of C/ebpb isoforms decreased SIM2s mRNA levels in C/ebpb immortalized mammary epithelial cell lines. These studies illustrate a novel pathway of tumor suppressor gene silencing in Ha-Ras-transformed breast epithelial cells and identify SIM2s as a target of C/EBPb and NOTCH signaling.

Activation of Notch1 in cells of the osteoblastic lineage inhibits osteoblast differentiation/function and causes osteopenia, whereas its activation in osteocytes causes a distinct osteopetrotic phenotype. To explore mechanisms responsible, we established the contributions of canonical Notch signaling (Rbpjκ dependent) to osteocyte function. Transgenics expressing Cre recombinase under the control of the dentin matrix protein-1 ( Dmp1) promoter were crossed with Rbpjκ conditional mice to generate Dmp1-Cre +/−; Rbpjκ Δ/Δ mice. These mice did not have a skeletal phenotype, indicating that Rbpjκ is dispensable for osteocyte function. To study the Rbpjκ contribution to Notch activation, Rosa Notch mice, where a loxP-flanked STOP cassette is placed between the Rosa26 promoter and the NICD coding sequence, were crossed with Dmp1-Cre transgenic mice and studied in the context ( Dmp1-Cre +/−; Rosa Notch; Rbpjκ Δ/Δ) or not ( Dmp1-Cre +/−; Rosa Notch) of Rbpjκ inactivation. Dmp1-Cre +/−; Rosa Notch mice exhibited increased femoral trabecular bone volume and decreased osteoclasts and bone resorption. The phenotype was reversed in the context of the Rbpjκ inactivation, demonstrating that Notch canonical signaling was accountable for the phenotype. Notch activation downregulated Sost and Dkk1 and upregulated Axin2, Tnfrsf11b, and Tnfsf11 mRNA expression, and these effects were not observed in the context of the Rbpjκ inactivation. In conclusion, Notch activation in osteocytes suppresses bone resorption and increases bone volume by utilization of canonical signals that also result in the inhibition of Sost and Dkk1 and upregulation of Wnt signaling.

Lateral meningocele syndrome (LMS), a genetic disorder characterized by meningoceles and skeletal abnormalities, is associated with NOTCH3 mutations. We created a mouse model of LMS (Notch3 tm1.1Ecan ) by introducing a tandem termination codon in the Notch3 locus upstream of the proline (P), glutamic acid (E), serine (S) and threonine (T) domain. Microcomputed tomography demonstrated that Notch3 tm1.1Ecan mice exhibit osteopenia. The cancellous bone osteopenia was no longer observed after the intraperitoneal administration of antibodies directed to the negative regulatory region (NRR) of Notch3. The anti-Notch3 NRR antibody suppressed the expression of Hes1, Hey1, and Hey2 (Notch target genes), and decreased Tnfsf11 (receptor activator of NF Kappa B ligand) messenger RNA in Notch3 tm1.1Ecan osteoblast (OB) cultures. Bone marrow-derived macrophages (BMMs) from Notch3 tm1.1Ecan

Notch receptors maintain skeletal homeostasis. NOTCH1 and 2 have been studied for their effects on bone remodeling. Although NOTCH3 plays a significant role in vascular physiology, knowledge about its function in other cellular environments, including bone, is limited. The present study was conducted to establish the function of NOTCH3 in skeletal cells using models of Notch3 misexpression. Microcomputed tomography demonstrated that Notch3 null mice did not have appreciable bone phenotypes. To study the effects of the NOTCH3 activation in the osteoblast lineage, BGLAP-Cre or Dmp1-Cre transgenics were crossed with Rosa Notch3 mice, where the NOTCH3 intracellular domain is expressed following the removal of a loxP -flanked STOP cassette. Microcomputed tomography demonstrated that BGLAP-Cre;Rosa Notch3 and Dmp1-Cre;Rosa Notch3 mice of both sexes exhibited an increase in trabecular bone and in connectivity, with a decrease in cortical bone and increased cortical porosity. Histological analysis revealed a decrease in osteoclast number and bone resorption in trabecular bone and an increase in osteoclast number and void or pore area in cortical bone of Rosa Notch3 mice. Bone formation was either decreased or could not be determined in Cre;Rosa Notch3 mice. NOTCH3 activation in osteoblasts inhibited Alpl (alkaline phosphatase) and Bglap (osteocalcin) and induced Tnfsf11 (RANKL) and Tnfrsf11b (osteoprotegerin) mRNA, possibly explaining the trabecular bone phenotype. However, NOTCH3 induced Tnfsf11 and suppressed Tnfrsf11b in osteocytes, possibly explaining the cortical porosity. In conclusion, basal NOTCH3 is dispensable for skeletal homeostasis, whereas activation of NOTCH3 in osteoblasts/osteocytes inhibits osteoclastogenesis and bone resorption in cancellous bone but increases intracortical remodeling and causes cortical porosity.

Nuclear factor of activated T cells (NFAT) c2 is important for the immune response and it compensates for NFATc1 for its effects on osteoclastogenesis, but its role in this process is not established. To study the function of NFATc2 in the skeleton, Nfatc2 mice, where the Nfact2 exon 2 is flanked by loxP sequences, were created and mated with mice expressing the Cre recombinase under the control of the Lyz2 promoter. Bone marrow-derived macrophage (BMM) from Lyz2 ;Nfatc2 mice cultured in the presence of macrophage-colony stimulating factor and receptor activator of NF-κB ligand exhibited a decrease in the number and size of osteoclasts and a smaller sealing zone when compared to BMMs from Nfatc2 littermate controls. Bone resorption was decreased in osteoclasts from Lyz2 ;Nfatc2 mice. This demonstrates that NFATc2 is necessary for optimal osteoclast maturation and function in vitro. Male and female Lyz2 ;Nfatc2 mice did not exhibit an obvious skeletal phenotype by microcomputed tomography (μCT) at either 1 or 4 months of age when compared to Nfatc2 sex-matched littermates. Bone histomorphometry confirmed the μCT results, and conditional 4-month-old Lyz2 ;Nfatc2 mice did not exhibit changes in parameters of bone histomorphometry. In conclusion, NFATc2 is necessary for optimal osteoclastogenesis in vitro, but its downregulation in the myeloid lineage has no consequences in skeletal remodeling in vivo.