New insights into the vitamin D/PTH axis in endocrine-driven metabolic bone diseases.

BACKGROUND: Endocrine regulation of bone metabolisms is the focus of the "Skeletal Endocrinology" series of meetings. AIMS: To report on the outcome of the discussion on the role of vitamin D/PTH axis in endocrine osteopathies held during the 10th Skeletal Endocrinology Meeting which took place in Stresa (Italy) in March 2023. OUTCOMES: Vitamin D/PTH axis has relevant influence on several outcomes in the general population and in patients affected by endocrinopathies such as hypoparathyroidism and secreting pituitary adenomas. CONCLUSIONS: Assessing the status of the vitamin D/PTH axis and using vitamin D and PTH as therapeutic agents is mandatory in several endocrine-related bone metabolic conditions.

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.

Antisense oligonucleotides targeting a NOTCH3 mutation in male mice ameliorate the cortical osteopenia of lateral meningocele syndrome.

Lateral Meningocele Syndrome (LMS) is a monogenic disorder associated with NOTCH3 pathogenic variants that result in the stabilization of NOTCH3 and a gain-of-function. A mouse model (Notch3em1Ecan) harboring a 6691-TAATGA mutation in the Notch3 locus that results in a functional outcome analogous to LMS exhibits cancellous and cortical bone osteopenia. We tested Notch3 antisense oligonucleotides (ASOs) specific to the Notch36691-TAATGA mutation for their effects on Notch3 downregulation and on the osteopenia of Notch3em1Ecan mice. Twenty-four mouse Notch3 mutant ASOs were designed and tested for toxic effects in vivo, and 12 safe ASOs were tested for their impact on the downregulation of Notch36691-TAATGA and Notch3 mRNA in osteoblast cultures from Notch3em1Ecan mice. Three ASOs downregulated Notch3 mutant transcripts specifically and were tested in vivo for their effects on the bone microarchitecture of Notch3em1Ecan mice. All three ASOs were well tolerated. One of these ASOs had more consistent effects in vivo and was studied in detail. The Notch3 mutant ASO downregulated Notch3 mutant transcripts in osteoblasts and bone marrow stromal cells and had no effect on other Notch receptors. The subcutaneous administration of Notch3 mutant ASO at 50 mg/Kg decreased Notch36691-TAATGA mRNA in bone without apparent toxicity; microcomputed tomography demonstrated that the ASO ameliorated the cortical osteopenia of Notch3em1Ecan mice but not the cancellous bone osteopenia. In conclusion, a Notch3 ASO that downregulates Notch3 mutant expression specifically ameliorates the cortical osteopenia in Notch3em1Ecan mice. ASOs may become useful strategies in the management of monogenic disorders affecting the skeleton.

Induced pluripotent stem cell technology in bone biology.

Technologies on the development and differentiation of human induced pluripotent stem cells (hiPSCs) are rapidly improving, and have been applied to create cell types relevant to the bone field. Differentiation protocols to form bona fide bone-forming cells from iPSCs are available, and can be used to probe details of differentiation and function in depth. When applied to iPSCs bearing disease-causing mutations, the pathogenetic mechanisms of diseases of the skeleton can be elucidated, along with the development of novel therapeutics. These cells can also be used for development of cell therapies for cell and tissue replacement.

Use of antisense oligonucleotides to target Notch3 in skeletal cells.

Notch receptors are determinants of cell fate and function, and play an important role in the regulation of bone development and skeletal remodeling. Lateral Meningocele Syndrome (LMS) is a monogenic disorder associated with NOTCH3 pathogenic variants that result in the stabilization of NOTCH3 and a gain-of-function. LMS presents with neurological developmental abnormalities and bone loss. We created a mouse model (Notch3em1Ecan) harboring a 6691TAATGA mutation in the Notch3 locus, and heterozygous Notch3em1Ecan mice exhibit cancellous and cortical bone osteopenia. In the present work, we explored whether Notch3 antisense oligonucleotides (ASO) downregulate Notch3 and have the potential to ameliorate the osteopenia of Notch3em1Ecan mice. Notch3 ASOs decreased the expression of Notch3 wild type and Notch36691-TAATGA mutant mRNA expressed by Notch3em1Ecan mice in osteoblast cultures without evidence of cellular toxicity. The effect was specific since ASOs did not downregulate Notch1, Notch2 or Notch4. The expression of Notch3 wild type and Notch36691-TAATGA mutant transcripts also was decreased in bone marrow stromal cells and osteocytes following exposure to Notch3 ASOs. In vivo, the subcutaneous administration of Notch3 ASOs at 25 to 50 mg/Kg decreased Notch3 mRNA in the liver, heart and bone. Microcomputed tomography demonstrated that the administration of Notch3 ASOs ameliorates the cortical osteopenia of Notch3em1Ecan mice, and ASOs decreased femoral cortical porosity and increased cortical thickness and bone volume. However, the administration of Notch3 ASOs did not ameliorate the cancellous bone osteopenia of Notchem1Ecan mice. In conclusion, Notch3 ASOs downregulate Notch3 expression in skeletal cells and their systemic administration ameliorates cortical osteopenia in Notch3em1Ecan mice; as such ASOs may become useful strategies in the management of skeletal diseases affected by Notch gain-of-function.

Skeletal disorders associated with the growth hormone-insulin-like growth factor 1 axis.

Growth hormone (GH) and insulin-like growth factor 1 (IGF1) are important regulators of bone remodelling and metabolism and have an essential role in the achievement and maintenance of bone mass throughout life. Evidence from animal models and human diseases shows that both GH deficiency (GHD) and excess are associated with changes in bone remodelling and cause profound alterations in bone microstructure. The consequence is an increased risk of fractures in individuals with GHD or acromegaly, a condition of GH excess. In addition, functional perturbations of the GH-IGF1 axis, encountered in individuals with anorexia nervosa and during ageing, result in skeletal fragility and osteoporosis. The effect of interventions used to treat GHD and acromegaly on the skeleton is variable and dependent on the duration of the disease, the pre-existing skeletal state, coexistent hormone alterations (such as those occurring in hypogonadism) and length of therapy. This variability could also reflect the irreversibility of the skeletal structural defect occurring during alterations of the GH-IGF1 axis. Moreover, the effects of the treatment of GHD and acromegaly on locally produced IGF1 and IGF binding proteins are uncertain and in need of further study. This Review highlights the pathophysiological, clinical and therapeutic aspects of skeletal fragility associated with perturbations in the GH-IGF1 axis.

Hairy and enhancer of split 1 is a primary effector of NOTCH2 signaling and induces osteoclast differentiation and function.

Notch2tm1.1Ecan mice, which harbor a mutation replicating that found in Hajdu-Cheney syndrome, exhibit marked osteopenia because of increased osteoclast number and bone resorption. Hairy and enhancer of split 1 (HES1) is a Notch target gene and a transcriptional modulator that determines osteoclast cell fate decisions. Transcript levels of Hes1 increase in Notch2tm1.1Ecan bone marrow-derived macrophages (BMMs) as they mature into osteoclasts, suggesting a role in osteoclastogenesis. To determine whether HES1 is responsible for the phenotype of Notch2tm1.1Ecan mice and the skeletal manifestations of Hajdu-Cheney syndrome, Hes1 was inactivated in Ctsk-expressing cells from Notch2tm1.1Ecan mice. Ctsk encodes the protease cathepsin K, which is expressed preferentially by osteoclasts. We found that the osteopenia of Notch2tm1.1Ecan mice was ameliorated, and the enhanced osteoclastogenesis was reversed in the context of the Hes1 inactivation. Microcomputed tomography revealed that the downregulation of Hes1 in Ctsk-expressing cells led to increased bone volume/total volume in female mice. In addition, cultures of BMMs from CtskCre/WT;Hes1Δ/Δ mice displayed a decrease in osteoclast number and size and decreased bone-resorbing capacity. Moreover, activation of HES1 in Ctsk-expressing cells led to osteopenia and enhanced osteoclast number, size, and bone resorptive capacity in BMM cultures. Osteoclast phenotypes and RNA-Seq of cells in which HES1 was activated revealed that HES1 modulates cell-cell fusion and bone-resorbing capacity by supporting sealing zone formation. In conclusion, we demonstrate that HES1 is mechanistically relevant to the skeletal manifestation of Notch2tm1.1Ecan mice and is a novel determinant of osteoclast differentiation and function.

Activation of Notch3 in osteoblasts/osteocytes causes compartment-specific changes in bone remodeling.

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 RosaNotch3 mice, where the NOTCH3 intracellular domain is expressed following the removal of a loxP-flanked STOP cassette. Microcomputed tomography demonstrated that BGLAP-Cre;RosaNotch3 and Dmp1-Cre;RosaNotch3 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 RosaNotch3 mice. Bone formation was either decreased or could not be determined in Cre;RosaNotch3 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.

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.

ENDOCRINOLOGY IN THE TIME OF COVID-19: Management of calcium metabolic disorders and osteoporosis.

Endocrinologists have had to make rapid changes to services so that resources can be focused on the COVID-19 response to help prevent spread of the virus. Herein we provide pragmatic advice on the management of commonly encountered calcium metabolic problems and osteoporosis. Non-urgent elective appointments should be postponed, and remote consultations and digital health solutions promoted. Patients should be empowered to self-manage their conditions safely. Patients, their caregivers and healthcare providers should be directed to assured national or international online resources and specific patient groups. For patients in acute hospital settings, existing emergency guidance on the management of hyper- and hypo-calcaemia should be followed. An approach to osteoporosis management is outlined. IV zoledronic acid infusions can be delayed for 6-9 months during the pandemic. Patients established on denosumab, teriparatide and abaloparatide should continue planned therapy. In the event of supply issues with teriparatide or abaloparatide, pausing this treatment in the short term is likely to be relatively harmless, whereas delaying denosumab may cause an immediate increased risk of fracture. The challenge of this pandemic will act as a catalyst to innovate within our management of metabolic bone and mineral disorders to ensure best use of resources and resilience of healthcare systems in its aftermath.

Nuclear factor of activated T cells 1 and 2 are required for vertebral homeostasis.

The present study defines the function of nuclear factor of activated T cells (NFAT)c1 and NFATc2 in osteoblast function in vivo and in vitro. Nfatc1loxP/loxP , Nfatc2loxP/loxP , and Nfatc1loxP/loxP ;Nfatc2loxP/loxP conditional mice were mated with BGLAP-Cre transgenics to inactivate Nfatc1 and Nfatc2 singly and in combination in osteoblasts. Microcomputed tomography demonstrated that male and female conditionally inactivated Nfatc1, Nfatc2 and dual Nfatc1;Nfatc2 mice had osteopenia at Lumbar 3 (L3) sites when compared to littermate controls. However, the Nfatc1 and Nfatc2 inactivation singly and in combination in Bglap-expressing osteoblasts did not result in an appreciable phenotype at femoral sites. Bone histomorphometry of L3 confirmed the osteopenic phenotype and demonstrated that Nfatc1;Nfatc2 inactivated male mice had a significant decrease in osteoblast number and in osteoblast surface and osteoid surface. The dual downregulation of Nfatc1 and Nfatc2 in bone marrow stromal cells caused a decrease in Alpl and Bglap expression, confirming a role of these transcription factors in osteoblast function. In conclusion, our studies reveal that NFATc1 and NFATc2 are necessary for optimal vertebral, but not femoral, bone homeostasis in vivo and osteoblast differentiation in vitro.

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.

The Skeleton of Lateral Meningocele Syndrome.

Notch (Notch1 through 4) are transmembrane receptors that determine cell differentiation and function, and are activated following interactions with ligands of the Jagged and Delta-like families. Notch has been established as a signaling pathway that plays a critical role in the differentiation and function of cells of the osteoblast and osteoclast lineages as well as in skeletal development and bone remodeling. Pathogenic variants of Notch receptors and their ligands are associated with a variety of genetic disorders presenting with significant craniofacial and skeletal manifestations. Lateral Meningocele Syndrome (LMS) is a rare genetic disorder characterized by neurological manifestations, meningoceles, skeletal developmental abnormalities and bone loss. LMS is associated with NOTCH3 gain-of-function pathogenic variants. Experimental mouse models of LMS revealed that the bone loss is secondary to increased osteoclastogenesis due to enhanced expression of receptor activator of nuclear factor kappa B ligand by cells of the osteoblast lineage. There are no effective therapies for LMS. Antisense oligonucleotides targeting Notch3 and antibodies that prevent the activation of NOTCH3 are being tested in preclinical models of the disease. In conclusion, LMS is a serious genetic disorder associated with NOTCH3 pathogenic variants. Novel experimental models have offered insight on mechanisms responsible and ways to correct the disease.

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.

Protease-Activated Receptor 1 Deletion Causes Enhanced Osteoclastogenesis in Response to Inflammatory Signals through a Notch2-Dependent Mechanism.

We found that protease-activated receptor 1 (PAR1) was transiently induced in cultured osteoclast precursor cells. Therefore, we examined the bone phenotype and response to resorptive stimuli of PAR1-deficient (knockout [KO]) mice. Bones and bone marrow-derived cells from PAR1 KO and wild-type (WT) mice were assessed using microcomputed tomography, histomorphometry, in vitro cultures, and RT-PCR. Osteoclastic responses to TNF-α (TNF) challenge in calvaria were analyzed with and without a specific neutralizing Ab to the Notch2-negative regulatory region (N2-NRR Ab). In vivo under homeostatic conditions, there were minimal differences in bone mass or bone cells between PAR1 KO and WT mice. However, PAR1 KO myeloid cells demonstrated enhanced osteoclastogenesis in response to receptor activator of NF-κB ligand (RANKL) or the combination of RANKL and TNF. Strikingly, in vivo osteoclastogenic responses of PAR1 KO mice to TNF were markedly enhanced. We found that N2-NRR Ab reduced TNF-induced osteoclastogenesis in PAR1 KO mice to WT levels without affecting WT responses. Similarly, in vitro N2-NRR Ab reduced RANKL-induced osteoclastogenesis in PAR1 KO cells to WT levels without altering WT responses. We conclude that PAR1 functions to limit Notch2 signaling in responses to RANKL and TNF and moderates osteoclastogenic response to these cytokines. This effect appears, at least in part, to be cell autonomous because enhanced osteoclastogenesis was seen in highly purified PAR1 KO osteoclast precursor cells. It is likely that this pathway is involved in regulating the response of bone to diseases associated with inflammatory signals.

MANAGEMENT OF ENDOCRINE DISEASE: Bone disorders associated with acromegaly: mechanisms and treatment.

Growth hormone (GH) and insulin-like growth factor-I (IGF-I) exert physiological actions on the skeleton throughout life, by stimulating longitudinal bone growth in children, the acquisition of bone mass during adolescence and the maintenance of skeletal architecture in adults. When GH and IGF-I are secreted in excess, bone remodeling is enhanced leading to deterioration of bone microstructure and impairment of bone strength. Indeed, acromegaly causes skeletal fragility, and vertebral fractures are reported in a remarkable number of subjects exposed to GH and IGF-I excess. The management of skeletal fragility in acromegaly is a challenge, since the awareness of this complication is low, the prediction of fracture risk is difficult to ascertain, the risk of fractures remains after the control of acromegaly and the effectiveness of bone-active drugs is unknown. This review is an update on bone disorders associated with acromegaly and provides a perspective of possible therapeutic approaches based on emerging pathophysiological and clinical information.

Excessive mechanical loading promotes osteoarthritis through the gremlin-1-NF-κB pathway.

Exposure of articular cartilage to excessive mechanical loading is deeply involved in the pathogenesis of osteoarthritis. Here, we identify gremlin-1 as a mechanical loading-inducible factor in chondrocytes, detected at high levels in middle and deep layers of cartilage after cyclic strain or hydrostatic pressure loading. Gremlin-1 activates nuclear factor-κB signalling, leading to subsequent induction of catabolic enzymes. In mice intra-articular administration of gremlin-1 antibody or chondrocyte-specific deletion of Gremlin-1 decelerates osteoarthritis development, while intra-articular administration of recombinant gremlin-1 exacerbates this process. Furthermore, ras-related C3 botulinum toxin substrate 1 activation induced by mechanical loading enhances reactive oxygen species (ROS) production. Amongst ROS-activating transcription factors, RelA/p65 induces Gremlin-1 transcription, which antagonizes induction of anabolic genes such as Sox9, Col2a1, and Acan by bone morphogenetic proteins. Thus, gremlin-1 plays essential roles in cartilage degeneration by excessive mechanical loading.

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.