NOTCH2 promotes osteoclast maturation and metabolism and modulates the transcriptome profile during osteoclastogenesis.

Notch signaling plays a key regulatory role in bone remodeling and NOTCH2 enhances osteoclastogenesis, an effect that is mostly mediated by its target gene Hes1. In the present study, we explored mechanisms responsible for the enhanced osteoclastogenesis in bone marrow-derived macrophages (BMM) from Notch2tm1.1Ecan, harboring a NOTCH2 gain-of-function mutation, and control mice. Notch2tm1.1Ecan mice are osteopenic and have enhanced osteoclastogenesis. Bulk RNA-Seq and gene set enrichment analysis of Notch2tm1.1Ecan BMMs cultured in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand revealed enrichment of genes associated with enhanced cell metabolism, aerobic respiration, and mitochondrial function, all associated with osteoclastogenesis. These pathways were not enhanced in the context of a Hes1 inactivation. Analysis of single cell RNA-Seq data of pooled control and Notch2tm1.1Ecan BMMs treated with M-CSF or M-CSF and receptor activator of NF-κB ligand for 3 days identified 11 well-defined cellular clusters. Pseudotime trajectory analysis indicated a trajectory of clusters expressing genes associated with osteoclast progenitors, osteoclast precursors, and mature cells. There were an increased number of cells expressing gene markers associated with the osteoclast and with an unknown, albeit related, cluster in Notch2tm1.1Ecan than in control BMMs as well as enhanced expression of genes associated with osteoclast progenitors and precursors in Notch2tm1.1Ecan cells. In conclusion, BMM cultures display cellular heterogeneity, and NOTCH2 enhances osteoclastogenesis, increases mitochondrial and metabolic activity of osteoclasts, and affects cell cluster allocation in BMMs.

NOTCH2 sensitizes the chondrocyte to the inflammatory response of tumor necrosis factor α.

Notch regulates the immune and inflammatory response and has been associated with the pathogenesis of osteoarthritis in humans and preclinical models of the disease. Notch2tm1.1Ecan mice harbor a NOTCH2 gain-of-function and are sensitized to osteoarthritis, but the mechanisms have not been explored. We examined the effects of tumor necrosis factor α (TNFα) in chondrocytes from Notch2tm1.1Ecan mice and found that NOTCH2 enhanced the effect of TNFα on Il6 and Il1b expression. Similar results were obtained in cells from a conditional model of NOTCH2 gain-of-function, Notch22.1Ecan mice, and following the expression of the NOTCH2 intracellular domain in vitro. Recombination signal-binding protein for immunoglobulin Kappa J region partners with the NOTCH2 intracellular domain to activate transcription; in the absence of Notch signaling it inhibits transcription, and Rbpj inactivation in chondrocytes resulted in Il6 induction. Although TNFα induced IL6 to a greater extent in the context of NOTCH2 activation, there was a concomitant inhibition of Notch target genes Hes1, Hey1, Hey2, and Heyl. Electrophoretic mobility shift assay demonstrated displacement of recombination signal-binding protein for immunoglobulin Kappa J region from DNA binding sites by TNFα explaining the increased Il6 expression and the concomitant decrease in Notch target genes. NOTCH2 enhanced the effect of TNFα on NF-κB signaling, and RNA-Seq revealed increased expression of pathways associated with inflammation and the phagosome in NOTCH2 overexpressing cells in the absence and presence of TNFα. Collectively, NOTCH2 has important interactions with TNFα resulting in the enhanced expression of Il6 and inflammatory pathways in chondrocytes.

Notch and the regulation of osteoclast differentiation and function.

Notch 1 through 4 are transmembrane receptors that play a pivotal role in cell differentiation and function; this review addresses the role of Notch signaling in osteoclastogenesis and bone resorption. Notch receptors are activated following interactions with their ligands of the Jagged and Delta-like families. In the skeleton, Notch signaling controls osteoclast differentiation and bone-resorbing activity either directly acting on osteoclast precursors, or indirectly acting on cells of the osteoblast lineage and cells of the immune system. NOTCH1 inhibits osteoclastogenesis, whereas NOTCH2 enhances osteoclast differentiation and function by direct and indirect mechanisms. NOTCH3 induces the expression of RANKL in osteoblasts and osteocytes and as a result induces osteoclast differentiation. There is limited expression of NOTCH4 in skeletal cells. Selected congenital disorders and skeletal malignancies are associated with dysregulated Notch signaling and enhanced bone resorption. In conclusion, Notch signaling is a critical pathway that controls osteoblast and osteoclast differentiation and function and regulates skeletal homeostasis in health and disease.

Antisense oligonucleotides targeting Notch2 ameliorate the osteopenic phenotype in a mouse model of Hajdu-Cheney syndrome.

Notch receptors play critical roles in cell-fate decisions and in the regulation of skeletal development and bone remodeling. Gain-of-function NOTCH2 mutations can cause Hajdu-Cheney syndrome, an untreatable disease characterized by osteoporosis and fractures, craniofacial developmental abnormalities, and acro-osteolysis. We have previously created a mouse model harboring a point 6955C→T mutation in the Notch2 locus upstream of the PEST domain, and we termed this model Notch2tm1.1Ecan Heterozygous Notch2tm1.1Ecan mutant mice exhibit severe cancellous and cortical bone osteopenia due to increased bone resorption. In this work, we demonstrate that the subcutaneous administration of Notch2 antisense oligonucleotides (ASO) down-regulates Notch2 and the Notch target genes Hes-related family basic helix-loop-helix transcription factor with YRPW motif 1 (Hey1), Hey2, and HeyL in skeletal tissue from Notch2tm1.1Ecan mice. Results of microcomputed tomography experiments indicated that the administration of Notch2 ASOs ameliorates the cancellous osteopenia of Notch2tm1.1Ecan mice, and bone histomorphometry analysis revealed decreased osteoclast numbers in Notch2 ASO-treated Notch2tm1.1Ecan mice. Notch2 ASOs decreased the induction of mRNA levels of TNF superfamily member 11 (Tnfsf11, encoding the osteoclastogenic protein RANKL) in cultured osteoblasts and osteocytes from Notch2tm1.1Ecan mice. Bone marrow-derived macrophage cultures from the Notch2tm1.1Ecan mice displayed enhanced osteoclastogenesis, which was suppressed by Notch2 ASOs. In conclusion, Notch2tm1.1Ecan mice exhibit cancellous bone osteopenia that can be ameliorated by systemic administration of Notch2 ASOs.

An antibody to Notch3 reverses the skeletal phenotype of lateral meningocele syndrome in male mice.

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 (Notch3tm1.1Ecan ) 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 Notch3tm1.1Ecan 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 Notch3tm1.1Ecan osteoblast (OB) cultures. Bone marrow-derived macrophages (BMMs) from Notch3tm1.1Ecan mutants exhibited enhanced osteoclastogenesis in culture, and this was increased in cocultures with Notch3tm1.1Ecan OB. Osteoclastogenesis was suppressed by anti-Notch3 NRR antibodies in Notch3tm1.1Ecan OB/BMM cocultures. In conclusion, the cancellous bone osteopenia of Notch3tm1.1Ecan mutants is reversed by anti-Notch3 NRR antibodies.

The Hajdu Cheney mutation sensitizes mice to the osteolytic actions of tumor necrosis factor α.

Hajdu Cheney syndrome (HCS) is characterized by craniofacial developmental abnormalities, acro-osteolysis, and osteoporosis and is associated with gain-of-NOTCH2 function mutations. A mouse model of HCS termed Notch2tm1.1Ecan harboring a mutation in exon 34 of Notch2 replicating the one found in HCS was used to determine whether the HCS mutation sensitizes the skeleton to the osteolytic effects of tumor necrosis factor α (TNFα). TNFα injected over the calvarial vault caused a greater increase in osteoclast number, osteoclast surface, and eroded surface in Notch2tm1.1Ecan mice compared with littermate WT controls. Accordingly, the effect of TNFα on osteoclastogenesis was greatly enhanced in cultures of bone marrow-derived macrophages (BMMs) from Notch2tm1.1Ecan mice when compared with the activity of TNFα in control cultures. TNFα induced the expression of Notch2 and Notch2 mutant mRNA by ∼2-fold, possibly amplifying the NOTCH2-dependent induction of osteoclastogenesis. The effect of TNFα on osteoclastogenesis in Notch2tm1.1Ecan mutants depended on NOTCH2 activation because it was reversed by anti-NOTCH2 negative regulatory region and anti-jagged 1 antibodies. The inactivation of Hes1 prevented the TNFα effect on osteoclastogenesis in the context of the Notch2tm1.1Ecan mutation. In addition, the induction of Il1b, but not of Tnfa and Il6, mRNA by TNFα was greater in Notch2tm1.1Ecan BMMs than in control cells, possibly contributing to the actions of TNFα and NOTCH2 on osteoclastogenesis. In conclusion, the HCS mutation enhances TNFα-induced osteoclastogenesis and the inflammatory bone-resorptive response possibly explaining the acro-osteolysis observed in affected individuals.

Cbl-PI3K interaction regulates Cathepsin K secretion in osteoclasts.

Effective bone resorption by osteoclasts is critical for balanced bone remodeling. We have previously reported that mice harboring a substitution mutation of tyrosine 737 to phenylalanine in the adapter protein Cbl (CblY737F, YF) have increased bone volume partly due to decreased osteoclast-mediated bone resorption. The CblY737F mutation abrogates interaction between Cbl and the p85 subunit of PI3K. Here, we studied the mechanism for defective resorptive function of YF mutant osteoclasts. The YF osteoclasts had intact actin cytoskeletons and sealing zones. Expression and localization of proteins needed for acidification of the resorptive lacunae were also comparable between the WT and YF osteoclasts. In contrast, secretion of Cathepsin K, a major protease needed to degrade collagen, was diminished in the conditioned media derived from YF osteoclasts. The targeting of Cathepsin K into LAMP2-positive vesicles was also compromised due to decreased number of LAMP2-positive vesicles in YF osteoclasts. Further, we found that in contrast to WT, conditioned media derived from YF osteoclasts promoted increased numbers of alkaline phosphatase positive colonies, and increased expression of osteogenic markers in WT calvarial cultures. Cumulatively, our results suggest that the Cbl-PI3K interaction regulates Cathepsin K secretion required for proper bone resorption, and secretion of factors which promote osteogenesis.

The lateral meningocele syndrome mutation causes marked osteopenia in mice.

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 (Notch3tm1.1Ecan ) by DNA sequencing of F1 mice. One-month-old male and female heterozygous Notch3tm1.1Ecan mice had cancellous and cortical bone osteopenia but exhibited no obvious neurological alterations, and histopathology of multiple organs revealed no abnormalities. Microcomputed tomography of these mutants revealed a 35-60% decrease in cancellous bone volume associated with a reduction in trabecular number and decreased connectivity. During maturation, cancellous and cortical bones were restored in female but not in male mice, which exhibited cancellous bone osteopenia at 4 months. Cancellous bone histomorphometry revealed increased osteoblast and osteocyte numbers and a modest increase in osteoclast surface and bone formation rate. Notch3tm1.1Ecan calvarial osteoblasts had increased proliferation and increased bone γ-carboxyglutamate protein (Bglap) and TNF superfamily member 11 (Tnfsf11) mRNA levels and lower Tnfrsf11b levels. Tnfsf11 mRNA was increased in osteocyte-rich femora from Notch3tm1.1Ecan mice. Cultures of bone marrow-derived macrophages from Notch3tm1.1Ecan mice revealed increased osteoclast formation, particularly in cocultures with osteoblasts from Notch3tm1.1Ecan mice. In conclusion, the Notch3tm1.1Ecan mutation causes osteopenia despite an increase in osteoblast proliferation and function and is associated with enhanced Tnfsf11 expression in osteoblasts and osteocytes.

The Hajdu Cheney Mutation Is a Determinant of B-Cell Allocation of the Splenic Marginal Zone.

The neurogenic locus notch homolog protein (Notch)-2 receptor is a determinant of B-cell allocation, and gain-of-NOTCH2-function mutations are associated with Hajdu-Cheney syndrome (HCS), a disease presenting with osteoporosis and acro-osteolysis. We generated a mouse model reproducing the HCS mutation (Notch2HCS), and heterozygous global mutant mice displayed gain-of-Notch2 function. In the mutant spleen, the characteristic perifollicular rim marking the marginal zone (MZ), which is the interface between the nonlymphoid red pulp and the lymphoid white pulp, merged with components of the white pulp. As a consequence, the MZ of Notch2HCS mice occupied most of the splenic structure. To explore the mechanisms involved, lymphocyte populations from the bone marrow and spleen were harvested from heterozygous Notch2HCS mice and sex-matched control littermates and analyzed by flow cytometry. Notch2HCS mice had an increase in CD21/35highCD23- splenic MZ B cells of approximately fivefold and a proportional decrease in splenic follicular B cells (CD21/35intCD23+) at 1, 2, and 12 months of age. Western blot analysis revealed that Notch2HCS mutant splenocytes had increased phospho-Akt and phospho-Jun N-terminal kinase, and gene expression analysis of splenic CD19+ B cells demonstrated induction of Hes1 and Hes5 in Notch2HCS mutants. Anti-Notch2 antibodies decreased MZ B cells in control and Notch2HCS mice. In conclusion, Notch2HCS mutant mice have increased mature B cells in the MZ of the spleen.

Sustained Notch2 signaling in osteoblasts, but not in osteoclasts, is linked to osteopenia in a mouse model of Hajdu-Cheney syndrome.

Individuals with Hajdu-Cheney syndrome (HCS) present with osteoporosis, and HCS is associated with NOTCH2 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 Notch2 mutations analogous to those in HCS (Notch2HCS) 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 (Tnfsf11) expression. Here, to determine the individual contributions of osteoclasts and osteoblasts to HCS osteopenia, we created a conditional-by-inversion (Notch2COIN ) model in which Cre recombination generates a Notch2ΔPEST allele expressing a Notch2 mutant lacking the PEST domain. Germ line Notch2COIN inversion phenocopied the Notch2HCS mutant, validating the model. To activate Notch2 in osteoclasts or osteoblasts, Notch2COIN mice were bred with mice expressing Cre from the Lyz2 or the BGLAP promoter, respectively. These crosses created experimental mice harboring a Notch2ΔPEST allele in Cre-expressing cells and control littermates expressing a wild-type Notch2 transcript. Notch2COIN inversion in Lyz2-expressing cells had no skeletal consequences and did not affect the capacity of bone marrow macrophages to form osteoclasts in vitro In contrast, Notch2COIN 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, Notch2 activation in osteoblast-enriched cultures from Notch2COIN mice induced Tnfsf11 expression. In conclusion, introduction of the HCS mutation in osteoblasts, but not in osteoclasts, causes osteopenia.

Loss of Cbl-PI3K interaction modulates the periosteal response to fracture by enhancing osteogenic commitment and differentiation.

The periosteum contains multipotent skeletal progenitors that contribute to bone repair. The signaling pathways regulating the response of periosteal cells to fracture are largely unknown. Phosphatidylinositol-3 Kinase (PI3K), a prominent lipid kinase, is a major signaling protein downstream of several factors that regulate osteoblast differentiation. Cbl is an E3 ubiquitin ligase and a major adaptor protein that binds to the p85 regulatory subunit and modulates PI3K activity. Substitution of tyrosine 737 to phenylalanine (Y737F) in Cbl abolishes the interaction between Cbl and p85 subunit without affecting the Cbl's ubiquitin ligase function. Here, we investigated the role of PI3K signaling during the very early stages of fracture healing using OsterixRFP reporter mice. We found that the absence of PI3K regulation by Cbl resulted in robust periosteal thickening, with increased proliferation of periosteal cells. While the multipotent properties of periosteal progenitors to differentiate into chondrocytes and adipocytes did not change, osteogenic differentiation in the absence of Cbl-PI3K interaction was highly augmented. The increased stability and nuclear localization of Osterix observed in periosteal cells lacking Cbl-PI3K interaction may explain this enhanced osteogenic differentiation since the expression of Osterix transcriptional target genes including osteocalcin and BSP are increased in YF cells. Overall, our findings highlight a hitherto unexplored and novel role for Cbl and PI3K in modulating the osteogenic response of periosteal cells during the early stages of fracture repair.

Interaction of Tumor Necrosis Factor Receptor-associated Factor 6 (TRAF6) and Vav3 in the Receptor Activator of Nuclear Factor κB (RANK) Signaling Complex Enhances Osteoclastogenesis.

The signaling pathway downstream of stimulation of receptor activator of nuclear factor κB (RANK) by RANK ligand is crucial for osteoclastogenesis. RANK recruits TNF receptor-associated factor 6 (TRAF6) to TRAF6-binding sites (T6BSs) in the RANK cytoplasmic tail (RANKcyto) to trigger downstream osteoclastogenic signaling cascades. RANKcyto harbors an additional highly conserved domain (HCR) that also activates crucial signaling during RANK-mediated osteoclastogenesis. However, the functional cross-talk between T6BSs and the HCR in the RANK signaling complex remains unclear. To characterize the cross-talk between T6BSs and the HCR, we screened TRAF6-interacting proteins using a proteomics approach. We identified Vav3 as a novel TRAF6 binding partner and evaluated the functional importance of the TRAF6-Vav3 interaction in the RANK signaling complex. We demonstrated that the coiled-coil domain of TRAF6 interacts directly with the Dbl homology domain of Vav3 to form the RANK signaling complex independent of the TRAF6 ubiquitination pathway. TRAF6 is recruited to the RANKcyto mutant, which lacks T6BSs, via the Vav3 interaction; conversely, Vav3 is recruited to the RANKcyto mutant, which lacks the IVVY motif, via the TRAF6 interaction. Finally, we determined that the TRAF6-Vav3 interaction resulting from cross-talk between T6BSs and the IVVY motif in RANKcyto enhances downstream NF-κB, MAPK, and NFATc1 activation by further strengthening TRAF6 signaling, thereby inducing RANK-mediated osteoclastogenesis. Thus, Vav3 is a novel TRAF6 interaction partner that functions in the activation of cooperative signaling between T6BSs and the IVVY motif in the RANK signaling complex.

Tumor necrosis factor (TNF) receptor-associated factor (TRAF)-interacting protein (TRIP) negatively regulates the TRAF2 ubiquitin-dependent pathway by suppressing the TRAF2-sphingosine 1-phosphate (S1P) interaction.

The signaling pathway downstream of TNF receptor (TNFR) is involved in the induction of a wide range of cellular processes, including cell proliferation, activation, differentiation, and apoptosis. TNFR-associated factor 2 (TRAF2) is a key adaptor molecule in TNFR signaling complexes that promotes downstream signaling cascades, such as nuclear factor-κB (NF-κB) and mitogen-activated protein kinase activation. TRAF-interacting protein (TRIP) is a known cellular binding partner of TRAF2 and inhibits TNF-induced NF-κB activation. Recent findings that TRIP plays a multifunctional role in antiviral response, cell proliferation, apoptosis, and embryonic development have increased our interest in exploring how TRIP can affect the TNFR-signaling pathway on a molecular level. In our current study, we demonstrated that TRIP is negatively involved in the TNF-induced inflammatory response through the down-regulation of proinflammatory cytokine production. Here, we demonstrated that the TRAF2-TRIP interaction inhibits Lys(63)-linked TRAF2 ubiquitination by inhibiting TRAF2 E3 ubiquitin (Ub) ligase activity. The TRAF2-TRIP interaction inhibited the binding of sphingosine 1-phosphate, which is a cofactor of TRAF2 E3 Ub ligase, to the TRAF2 RING domain. Finally, we demonstrated that TRIP functions as a negative regulator of proinflammatory cytokine production by inhibiting TNF-induced NF-κB activation. These results indicate that TRIP is an important cellular regulator of the TNF-induced inflammatory response.

D-chiro-inositol negatively regulates the formation of multinucleated osteoclasts by down-regulating NFATc1.

PURPOSE: Osteoclasts (OCs) are multinucleated giant cells that resorb bone matrix. Accelerated bone destruction by OCs might cause several metabolic bone-related diseases, such as osteoporosis and inflammatory bone loss. D-pinitol (3-O-methyl-D-chiro-inositol) is a prominent component of dietary legumes and is actively converted to D-chiro-inositol, which is a putative insulin-like mediator. In this study, we analyzed the effect of D-chiro-inositol on OC differentiation. METHODS: To analyze the role of D-chiro-inositol on OC differentiation, we examined OC differentiation by the three types of osteoclastogenesis cultures with tartrate-resistant acid phosphatase (TRAP) staining and solution assay. Then, we carried out cell fusion assay with purified TRAP(+) mononuclear OC precursors. Finally, we analyzed the effect of D-chiro-inositol on OC maker expression in response to the regulation of nuclear factor of activated T cells c1 (NFATc1). RESULTS: We demonstrated that D-chiro-inositol acts as an inhibitor of receptor activator of NF-κB ligand-induced OC differentiation. The formation of multinucleated OCs by cell-cell fusion is reduced by treatment with D-chiro-inositol in a dose-dependent manner. In addition, we demonstrated that D-chiro-inositol inhibits the expression of several osteoclastogenic genes by down-regulating NFATc1. CONCLUSIONS: We have shown that D-chiro-inositol is negatively involved in osteoclastogenesis through the inhibition of multinucleated OC formation by cell-cell fusion. The expression of NFATc1 was significantly down-regulated by D-chiro-inositol in OCs and consequently, the expression of OC marker genes was significantly reduced. Hence, these results show that D-chiro-inositol might be a good candidate to treat inflammatory bone-related diseases or secondary osteoporosis in diabetes mellitus.