Multi-omics analyses reveal aberrant differentiation trajectory with WNT1 loss-of-function in type XV osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a group of severe genetic bone disorders characterized by congenital low bone mass, deformity and frequent fractures. Type XV OI is a moderate to severe form of skeletal dysplasia caused by WNT1 variants. In this cohort study from southern China, we summarized the clinical phenotypes of patients with WNT1 variants and found that the proportion of type XV patients was around 10.3% (25 out of 243) with a diverse spectrum of phenotypes. Functional assays indicated that variants of WNT1 significantly impaired its secretion and effective activity, leading to moderate to severe clinical manifestations, porous bone structure and enhanced osteoclastic activities. Analysis of proteomic data from human skeleton indicated that the expression of SOST was dramatically reduced in type XV patients when comparing to the patients with COL1A1 quantitative variants. Single-cell transcriptome data generated from the human tibia samples of patients diagnosed with type XV OI and leg-length-discrepancy respectively, revealed aberrant differentiation trajectory of skeletal progenitors and impaired maturation of osteocytes with loss of WNT1, resulting in excessive CXCL12+ progenitors, fewer mature osteocytes and existence of abnormal cell populations with adipogenic characteristics. The integration of multi-omics data from human skeleton delineates how WNT1 regulates the differentiation and maturation of skeletal progenitors, which will provide a new direction for the treatment strategy of type XV osteogenesis imperfecta and relative low bone mass diseases such as early onset osteoporosis.

Osteogenesis imperfecta is a rare disease characterized by low bone mass, frequent fractures and long bone deformity. Type XV osteogenesis imperfect is an autosomal recessive disorder caused by WNT1 variants, while heterozygous variants of WNT1 result in early onset osteoporosis. In this cohort study, we summarized the clinical features of 25 patients diagnosed with type XV osteogenesis imperfect. The WNT1 variants were confirmed by genetic test. Molecular assays were conducted to reveal the impact of variants on WNT1 protein activity and bone structure. We then compared the protein levels in bone tissues isolated from the type XV patients and patients with mild deformity using proteomic method, and found the expression of SOST, mainly produced by mature osteoblasts and osteocytes, was dramatically reduced in type XV patients. We further compared the global mRNA expression levels in the skeletal cells using single-cell RNA sequencing. Analyses of these data indicated that more immature progenitors were identified and maturation of osteocytes was impaired with WNT1 loss-of-function. Our study helps to understand the underlying pathogenesis of type XV osteogenesis imperfecta.

Calorie restriction in mice impairs cortical but not trabecular peak bone mass by suppressing bone remodeling.

Calorie restriction (CR) can lead to weight loss and decreased substrate availability for bone cells. Ultimately, this can lead to impaired peak bone acquisition in children and adolescence and bone loss in adults. But the mechanisms that drive diet-induced bone loss in humans are not well characterized. To explore those in greater detail, we examined the impact of 30% CR for 4 and 8 wk in both male and female 8-wk-old C57BL/6 J mice. Body composition, areal bone mineral density (aBMD), skeletal microarchitecture by micro-CT, histomorphometric parameters, and in vitro trajectories of osteoblast and adipocyte differentiation were examined. After 8 wk, CR mice lost weight and exhibited lower femoral and whole-body aBMD vs ad libitum (AL) mice. By micro-CT, CR mice had lower cortical bone area fraction vs AL mice, but males had preserved trabecular bone parameters and females showed increased bone volume fraction compared to AL mice. Histomorphometric analysis revealed that CR mice had a profound suppression in trabecular as well as endocortical and periosteal bone formation in addition to reduced bone resorption compared to AL mice. Bone marrow adipose tissue was significantly increased in CR mice. In vitro, the pace of adipogenesis in bone marrow stem cells was greatly accelerated with higher markers of adipocyte differentiation and more oil red O staining, whereas osteogenic differentiation was reduced. qRT-PCR and western blotting suggested that the expression of Wnt16 and the canonical ß-catenin pathway was compromised during CR. In sum, CR causes impaired peak cortical bone mass due to a profound suppression in bone remodeling. The increase in marrow adipocytes in vitro and in vivo is related to both progenitor recruitment and adipogenesis in the face of nutrient insufficiency. Long-term CR may lead to lower bone mass principally in the cortical envelope, possibly due to impaired Wnt signaling.

Calorie restriction led to impaired bone mass and increased accumulation of bone marrow adipose tissue. During the development of bone-fat imbalance due to calorie restriction, bone remodeling was notably inhibited. Calorie restriction may shift the differentiation of bone marrow stem cells toward adipocytes instead of osteoblasts. This process involves a disruption in the canonical Wnt signaling pathway.

Are Osteoblasts Multiple Cell Types? A New Diversity in Skeletal Stem Cells and Their Derivatives.

Only in the past decade have skeletal stem cells (SSCs), a cell type displaying formal evidence of stemness and serving as the ultimate origin of mature skeletal cell types such as osteoblasts, been defined. Here, we discuss a pair of recent reports that identify that SSCs do not represent a single cell type, but rather a family of related cells that each have characteristic anatomic locations and distinct functions tailored to the physiology of those sites. The distinct functional properties of these SSCs in turn provide a basis for the diseases of their respective locations. This concept emerges from one report identifying a distinct vertebral skeletal stem cell driving the high rate of breast cancer metastasis to the spine over other skeletal sites and a report identifying two SSCs in the calvaria that interact to mediate both physiologic calvarial mineralization and pathologic calvarial suture fusion in craniosynostosis. Despite displaying functional differences, these SSCs are each united by shared features including a shared series of surface markers and parallel differentiation hierarchies. We propose that this diversity at the level of SSCs in turn translates into a similar diversity at the level of mature skeletal cell types, including osteoblasts, with osteoblasts derived from different SSCs each displaying different functional and transcriptional characteristics reflecting their cell of origin. In this model, osteoblasts would represent not a single cell type, but rather a family of related cells each with distinct functions, paralleling the functional diversity in SSCs.

Only in the past decade have the stem cells in the skeleton been identified. Here, we discuss a pair of recent reports that identify that skeletal stem cells are actually a family of related cells that each have distinct locations and functions. These site-specific skeletal stem cells account for the signature diseases occurring in different regions of the skeleton. Specifically, one of these stem cells forms the spine and establishes that this stem cell drives the high rate of breast cancer metastasis to the spine over other skeletal sites. There are also at least two skeletal stem cells in the flat bones of the skull, with mutations alerting how these two stem cells "talk" to each other serving as a cause for disorders of premature skull fusion. Despite displaying differences in their function, these stem cells are each united by shared features including a partially shared series marker genes. We also here propose that this diversity at the level of skeletal stem cells translates into a similar diversity in mature skeletal cell types, including osteoblasts. In this model, osteoblasts are not a single cell type, but rather a family of related cells each with distinct functions.

Evaluation of Romosozumab's Effects on Bone Marrow Adiposity in Postmenopausal Osteoporotic Women: Results from the FRAME Bone Biopsy Sub-Study.

Romosozumab, a humanized monoclonal antibody that binds and inhibits sclerostin produces a marked increase in bone formation with a concomitant decreased bone resorption. This transient rise in bone formation in the first two months of treatment is mainly due to an increased modeling-based bone formation. This requires the recruitment and differentiation of osteoblasts, one possibility being a preferential switch in commitment of precursors to osteoblasts over adipocytes. The purpose of this study was to analyze the marrow adiposity in transiliac bone biopsies at months 2 or 12 from the FRAME biopsy sub-study in patients receiving romosozumab or placebo. The total adipocyte area, number and density were measured on the total cancellous bone area. The size and shape at the individual adipocyte level were assessed including the mean adipocyte area, perimeter, min and max diameters and aspect ratio. No significant difference in total adipocyte area, number or density between placebo and romosozumab groups was observed at months 2 and 12, and no difference was observed between 2 and 12 months. After 2 or 12 months, romosozumab did not modify the size or shape of the adipocytes. No relationship between the adipocyte parameters and the dynamic parameters of bone formation could be evidenced. In conclusion, based on the analysis of a small number of biopsies, no effect of romosozumab on bone marrow adiposity of iliac crest was identified after 2 and 12 months suggesting that the modeling-based formation observed at month 2 was not due to a preferential commitment of the precursor to osteoblast over adipocyte cell lines but may result from a reactivation of bone lining cells and from a progenitor pool independent of the marrow adipocyte population.

Osteoporosis is characterized by bone loss resulting from an imbalance between the bone resorption and the bone formation in favor of the resorption. Romosozumab, a new medication to treat osteoporosis, has been shown to induce an early transient increase in bone formation that requires the differentiation of new bone forming cells called osteoblasts. Osteoblasts and fat-containing cells known as adipocytes present in the bone marrow originate from a common precursor cell. Thus, a preferential switch of this precursor to osteoblast over adipocyte is thought to be a possible cause for the increase in bone formation. The purpose of this study was to analyze the bone marrow adipocytes on bone biopsies from the pelvis in osteoporotic patients treated with romosozumab in order to evaluate that possibility. After treatment, the proportion of adipocytes, their size and shape, did not change when compared to untreated patients. In conclusion, no effect of romosozumab on bone marrow adipocytes was identified suggesting that the increased bone formation induced by romosozumab was not due to a preferential differentiation of precursor cells to osteoblasts over adipocytes.

CHD7 regulates craniofacial cartilage development via controlling HTR2B expression.

Mutations in the Chromodomain helicase DNA-binding protein 7 - coding gene (CHD7) cause CHARGE syndrome (CS). Although craniofacial and skeletal abnormalities are major features of CS patients, the role of CHD7 in bone and cartilage development remain largely unexplored. Here, using a zebrafish (Danio rerio) CS model, we show that chd7-/- larvae display abnormal craniofacial cartilage development and spinal deformities. The craniofacial and spine defects are accompanied by a marked reduction of bone mineralization. At the molecular level, we show that these phenotypes are associated with significant reduction in the expression levels of osteoblast differentiation markers. Additionally, we detected a marked depletion of collagen 2α1 in the cartilage of craniofacial regions and vertebrae, along with significantly reduced number of chondrocytes. Chondrogenesis defects are at least in part due to downregulation of htr2b, which we found to be also dysregulated in human cells derived from an individual with CHD7 mutation-positive CS. Overall, this study thus unveils an essential role for CHD7 in cartilage and bone development, with potential clinical relevance for the craniofacial defects associated with CS.

Patients with CHARGE syndrome exhibit skeletal defects. CHARGE syndrome is primarily caused by mutations in the chromatin remodeler-coding gene CHD7. To investigate the poorly characterized role of CHD7 in cartilage and bone development, here, we examine the craniofacial and bone anomalies in a zebrafish chd7-/- mutant model. We find that zebrafish mutant larvae exhibit striking dysmorphism of craniofacial structures and spinal deformities. Notably, we find a significant reduction in osteoblast, chondrocyte, and collagen matrix markers. This work provides important insights to improve our understanding of the role of chd7 in skeletal development.

Sphingosine-1-phosphate promotes osteogenesis by stimulating osteoblast growth and neovascularization in a vascular endothelial growth factor-dependent manner.

Sphingosine-1-phosphate (S1P) plays multiple roles in bone metabolism and regeneration. Here, we have identified a novel S1P-regulated osteoanabolic mechanism functionally connecting osteoblasts (OBs) to the highly specialized bone vasculature. We demonstrate that S1P/S1PR3 signaling in OBs stimulates vascular endothelial growth factor a (VEGFa) expression and secretion to promote bone growth in an autocrine and boost osteogenic H-type differentiation of bone marrow endothelial cells in a paracrine manner. VEGFa-neutralizing antibodies and VEGF receptor inhibition by axitinib abrogated OB growth in vitro and bone formation in male C57BL/6J in vivo following S1P stimulation and S1P lyase inhibition, respectively. Pharmacological S1PR3 inhibition and genetic S1PR3 deficiency suppressed VEGFa production, OB growth in vitro, and inhibited H-type angiogenesis and bone growth in male mice in vivo. Together with previous work on the osteoanabolic functions of S1PR2 and S1PR3, our data suggest that S1P-dependent bone regeneration employs several nonredundant positive feedback loops between OBs and the bone vasculature. The identification of this yet unappreciated aspect of osteoanabolic S1P signaling may have implications for regular bone homeostasis as well as diseases where the bone microvasculature is affected such as age-related osteopenia and posttraumatic bone regeneration.

Sphingosine-1-phosphate (S1P) is a signaling lipid that regulates bone growth and regeneration. In the present study, a novel regenerative mechanism was connected to S1P signaling within the bone. Activation of its receptor S1PR3 in bone-forming osteoblasts led to secretion of vascular endothelial growth factor a (VEGFa), the most potent vessel-stimulating factor. This stimulated the development of specialized vessels of the bone marrow, the H-type vessels, that supported overall bone regeneration. These findings foster our understanding of regular bone metabolism and suggest that S1P-based drugs may help treat diseases such as age-related osteopenia and posttraumatic bone regeneration, conditions crucially dependent on functional bone microvasculature.

Phlpp1 Expression in Osteoblasts Plays a Modest Role in Bone Homeostasis.

Prior work demonstrated that Phlpp1 deficiency alters limb length and bone mass, but the cell types involved and requirement of Phlpp1 for this effect were unclear. To understand the function of Phlpp1 within bone-forming osteoblasts, we crossed Phlpp1 floxed mice with mice harboring type 1 collagen (Col1a12.3kb)-Cre. Mineralization of bone marrow stromal cell cultures derived from Phlpp1 cKOCol1a1 was unchanged, but levels of inflammatory genes (eg, Ifng, Il6, Ccl8) and receptor activator of NF-κB ligand/osteoprotegerin (RANKL/OPG) ratios were enhanced by either Phlpp1 ablation or chemical inhibition. Micro-computed tomography of the distal femur and L5 vertebral body of 12-week-old mice revealed no alteration in bone volume per total volume, but compromised femoral bone microarchitecture within Phlpp1 cKOCol1a1 conditional knockout females. Bone histomorphometry of the proximal tibia documented no changes in osteoblast or osteoclast number per bone surface but slight reductions in osteoclast surface per bone surface. Overall, our data show that deletion of Phlpp1 in type 1 collagen-expressing cells does not significantly alter attainment of peak bone mass of either males or females, but may enhance inflammatory gene expression and the ratio of RANKL/OPG. Future studies examining the role of Phlpp1 within models of advanced age, inflammation, or osteocytes, as well as functional redundancy with the related Phlpp2 isoform are warranted. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Microvascular Disease Associates with Larger Osteocyte Lacunae in Cortical Bone in Type 2 Diabetes Mellitus.

Clinical studies indicate that microvascular disease (MVD) affects bone microstructure and decreases bone strength in type 2 diabetes mellitus (T2D). Osteocytes are housed in small voids within the bone matrix and lacunae and act as sensors of mechanical forces in bone. These cells regulate osteoclastic bone resorption and osteoblastic bone formation as well as osteocytic perilacunar remodeling. We hypothesized that MVD changes morphometric osteocyte lacunar parameters in individuals with T2D. We collected iliac crest bone biopsies from 35 individuals (10 female, 25 male) with T2D with MVD (15%) or without MVD (21%) with a median age of 67 years (interquartile range [IQR] 62-72 years). The participants were included based on c-peptide levels >700 pmol L-1, absence of anti-GAD65 antibodies, and glycated hemoglobin (HbA1c) levels between 40 and 82 mmol mol-1 or 5.8% and 9.7%, respectively. We assessed osteocyte lacunar morphometric parameters in trabecular and cortical bone regions using micro-computed tomography (micro-CT) at a nominal resolution of 1.2 µm voxel size. The cortical osteocyte lacunar volume (Lc.V) was 7.7% larger (p = 0.05) and more spherical (Lc.Sr, p < 0.01) in the T2D + MVD group. Using linear regression, we found that lacunar density (Lc.N/BV) in trabecular but not cortical bone was associated with HbA1c (p < 0.05, R 2 = 0.067) independently of MVD. Furthermore, Lc.V was larger and Lc.Sr higher in the center than in the periphery of the trabecular and cortical bone regions (p < 0.05). In conclusion, these data imply that MVD may impair skeletal integrity, possibly contributing to increased skeletal fragility in T2D complicated by MVD. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Efficacy and Toxicity of Calcitonin Treatment in Children with Cherubism: A Single-Center Cohort Study.

Cherubism is a rare autosomal dominant disease characterized by expansile osteolytic jawbone lesions. The effect and safety of off-label calcitonin treatment during the progressive phase of the disease are not well described. In this retrospective study, we present data on the radiological response and adverse effects of subcutaneously administered calcitonin in a cohort of nine cherubism children (three female, six male). Two of the nine patients underwent two separate treatment courses with a significant off-treatment interval in between; therefore, a total of 11 treatment courses with a mean duration of 17.9 months (range <1 to 35, SD 10.8) were studied. To measure the response, the cumulative volume of cherubism lesions was calculated from available three-dimensional imaging. The primary outcome was the change in the volume of lesions during calcitonin treatment and only assessed for the eight treatment courses with a minimal duration of 6 months. A statistically significant reduction in the mean cumulative volume of lesions was seen regardless of treatment duration. Average volume reduction was highest in the first half year of treatment, with a gradual, ongoing reduction thereafter. For the secondary outcome, the change in the cumulative volume of lesions after treatment cessation was assessed for the seven treatment courses with follow-up imaging available. After six of these seven treatment courses, the cumulative volume increased again but remained undoubtedly smaller than the initial volume at the start of therapy. Adverse effects were assessed for all 11 treatment courses and occurred in 73% of them. Most adverse effects were mild and low grade, with the most severe being one grade 3 symptomatic hypocalcemia requiring hospitalization and early treatment termination. Calcitonin treatment seems effective and tolerable in treating actively progressing cherubism in children. However, further research is required to better understand the pharmacological treatment of cherubism, including also other drugs, dosing, and protocols. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

GSTT1 as a Predictive Marker and Enhancer for Osteogenic Potential of Human Adipose-Derived Stromal/Stem Cells.

Adipose-derived stromal/stem cells (ASCs) have been extensively studied as cell sources for regenerative medicine for bone because of their excellent proliferative capacity and the ability to obtain a large number of cells with minimal donor morbidity. On the other hand, the differentiation potential of ASCs is generally lower than that of bone marrow-derived stromal/stem cells and varies greatly depending on donors. In this study, we mined a marker that can predict the osteogenic potential of ASC clones and also investigated the usefulness of the molecule as the enhancer of osteogenic differentiation of ASCs as well as its mechanism of action. Through RNA-seq gene analysis, we discovered that GSTT1 (Glutathione S-transferase theta-1) was the most distinguished gene marker between highly osteogenic and poorly osteogenic ASC clones. Knockdown of GSTT1 in high osteogenic ASCs by siGSTT1 treatment reduced mineralized matrix formation. On the other hand, GSTT1 overexpression by GSTT1 transfection or GSTT1 recombinant protein treatment enhanced osteogenic differentiation of low osteogenic ASCs. Metabolomic analysis confirmed significant changes of metabolites related to bone differentiation in ASCs transfected with GSTT1. A high total antioxidant capacity, low levels of cellular reactive oxygen species, and increased GSH/GSSG ratios were also detected in GSTT1-transfected ASCs. When the in vivo effect of GSTT1-transfected ASCs on bone regeneration was investigated with segmental long-bone defect model in rats, bone regeneration was significantly better after implantation of GSTT1-transfected ASCs compared with that of control vector-transfected ASCs. In conclusion, GSTT1 can be a useful marker to screen the highly osteogenic ASC clones and also a therapeutic factor to enhance the osteogenic differentiation of poorly osteogenic ASC clones. © 2023 American Society for Bone and Mineral Research (ASBMR).

L-BAIBA Synergizes with Sub-Optimal Mechanical Loading to Promote New Bone Formation.

The L-enantiomer of ß-aminoisobutyric acid (BAIBA) is secreted by contracted muscle in mice, and exercise increases serum levels in humans. In mice, L-BAIBA reduces bone loss with unloading, but whether it can have a positive effect with loading is unknown. Since synergism can be more easily observed with sub-optimal amounts of factors/stimulation, we sought to determine whether L-BAIBA could potentiate the effects of sub-optimal loading to enhance bone formation. L-BAIBA was provided in drinking water to C57Bl/6 male mice subjected to either 7 N or 8.25 N of sub-optimal unilateral tibial loading for 2 weeks. The combination of 8.25 N and L-BAIBA significantly increased the periosteal mineral apposition rate and bone formation rate compared to loading alone or BAIBA alone. Though L-BAIBA alone had no effect on bone formation, grip strength was increased, suggesting a positive effect on muscle function. Gene expression analysis of the osteocyte-enriched bone showed that the combination of L-BAIBA and 8.25 N induced the expression of loading-responsive genes such as Wnt1, Wnt10b, and the TGFb and BMP signaling pathways. One dramatic change was the downregulation of histone genes in response to sub-optimal loading and/or L-BAIBA. To determine early gene expression, the osteocyte fraction was harvested within 24 hours of loading. A dramatic effect was observed with L-BAIBA and 8.25 N loading as genes were enriched for pathways regulating the extracellular matrix (Chad, Acan, Col9a2), ion channel activity (Scn4b, Scn7a, Cacna1i), and lipid metabolism (Plin1, Plin4, Cidec). Few changes in gene expression were observed with sub-optimal loading or L-BAIBA alone after 24 hours. These results suggest that these signaling pathways are responsible for the synergistic effects between L-BAIBA and sub-optimal loading. Showing that a small muscle factor can enhance the effects of sub-optimal loading of bone may be of relevance for individuals unable to benefit from optimal exercise. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

MicroRNA-19a-3p Decreases with Age in Mice and Humans and Inhibits Osteoblast Senescence.

Aging is a major risk factor for most chronic diseases, including osteoporosis, and is characterized by an accumulation of senescent cells in various tissues. MicroRNAs (miRNAs) are critical regulators of bone aging and cellular senescence. Here, we report that miR-19a-3p decreases with age in bone samples from mice as well as in posterior iliac crest bone biopsies of younger versus older healthy women. miR-19a-3p also decreased in mouse bone marrow stromal cells following induction of senescence using etoposide, H2O2, or serial passaging. To explore the transcriptomic effects of miR-19a-3p, we performed RNA sequencing of mouse calvarial osteoblasts transfected with control or miR-19a-3p mimics and found that miR-19a-3p overexpression significantly altered the expression of various senescence, senescence-associated secretory phenotype-related, and proliferation genes. Specifically, miR-19a-3p overexpression in nonsenescent osteoblasts significantly suppressed p16 Ink4a and p21 Cip1 gene expression and increased their proliferative capacity. Finally, we established a novel senotherapeutic role for this miRNA by treating miR-19a-3p expressing cells with H2O2 to induce senescence. Interestingly, these cells exhibited lower p16 Ink4a and p21 Cip1 expression, increased proliferation-related gene expression, and reduced SA-ß-Gal+ cells. Our results thus establish that miR-19a-3p is a senescence-associated miRNA that decreases with age in mouse and human bones and is a potential senotherapeutic target for age-related bone loss. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Molecular Dissection of Somatic Skeletal Disease in Neurofibromatosis Type 1.

Neurofibromatosis type 1 (NF1) is a tumor predisposition syndrome caused by heterozygous NF1 gene mutations. Patients with NF1 present with pleiotropic somatic secondary manifestations, including development of bone pseudarthrosis after fracture. Somatic NF1 gene mutations were reproducibly identified in patient-derived pseudarthrosis specimens, suggesting a local mosaic cell population including somatic pathologic cells. The somatic cellular pathogenesis of NF1 pseudarthroses remains unclear, though defects in osteogenesis have been posited. Here, we applied time-series single-cell RNA-sequencing (scRNA-seq) to patient-matched control and pseudarthrosis-derived primary bone stromal cells (BSCs). We show that osteogenic specification to an osteoblast progenitor cell population was evident for control bone-derived cells and haploinsufficient pseudarthrosis-derived cells. Similar results were observed for somatic patient fracture-derived NF1-/- cells; however, expression of genetic pathways associated with skeletal mineralization were significantly reduced in NF1-/- cells compared with fracture-derived NF1+/- cells. In mice, we show that Nf1 expressed in bone marrow osteoprogenitors is required for the maintenance of the adult skeleton. Results from our study implicate impaired Clec11a-Itga11-Wnt signaling in the pathogenesis of NF1-associated skeletal disease. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Similarities Between Disuse and Age-Induced Bone Loss.

Disuse and aging are known risk factors associated with low bone mass and quality deterioration, resulting in increased fracture risk. Indeed, current and emerging evidence implicate a large number of shared skeletal manifestations between disuse and aging scenarios. This review provides a detailed overview of current preclinical models of musculoskeletal disuse and the clinical scenarios they seek to recapitulate. We also explore and summarize the major similarities between bone loss after extreme disuse and advanced aging at multiple length scales, including at the organ/tissue, cellular, and molecular level. Specifically, shared structural and material alterations of bone loss are presented between disuse and aging, including preferential loss of bone at cancellous sites, cortical thinning, and loss of bone strength due to enhanced fragility. At the cellular level bone loss is accompanied, during disuse and aging, by increased bone resorption, decreased formation, and enhanced adipogenesis due to altered gap junction intercellular communication, WNT/ß-catenin and RANKL/OPG signaling. Major differences between extreme short-term disuse and aging are discussed, including anatomical specificity, differences in bone turnover rates, periosteal modeling, and the influence of subject sex and genetic variability. The examination also identifies potential shared mechanisms underlying bone loss in aging and disuse that warrant further study such as collagen cross-linking, advanced glycation end products/receptor for advanced glycation end products (AGE-RAGE) signaling, reactive oxygen species (ROS) and nuclear factor κB (NF-κB) signaling, cellular senescence, and altered lacunar-canalicular connectivity (mechanosensation). Understanding the shared structural alterations, changes in bone cell function, and molecular mechanisms common to both extreme disuse and aging are paramount to discovering therapies to combat both age-related and disuse-induced osteoporosis. © 2022 American Society for Bone and Mineral Research (ASBMR).

Cyclic Adenosine Monophosphate (cAMP)-Dependent Phosphodiesterase Inhibition Promotes Bone Anabolism Through CD8+ T Cell Wnt-10b Production in Mice.

Cyclic adenosine monophosphate (cAMP)-dependent phosphodiesterase (PDE) inhibitors such as pentoxifylline (PTX) suppress cAMP degradation and promote cAMP-dependent signal transduction. PDE inhibitors increase bone formation and bone mass in preclinical models and are used clinically to treat psoriatic arthritis by targeting inflammatory mediators including activated T cells. T cell activation requires two signals: antigen-dependent CD3-activation, which stimulates cAMP production; and CD28 co-stimulation, which downregulates cAMP-signaling, through PDE activation. PDE-inhibitors consequently suppress T cell activation by disrupting CD28 co-stimulation. Interestingly, we have reported that when CD8+ T cells are activated in the absence of CD28 co-stimulation, they secrete Wnt-10b, a bone anabolic Wnt ligand that promotes bone formation. In the present study, we investigated whether the bone anabolic activity of the PDE-inhibitor PTX, has an immunocentric basis, involving Wnt-10b production by CD8+ T cells. When wild-type (WT) mice were administered PTX, biochemical markers of both bone resorption and formation were significantly increased, with net bone gain in the axial skeleton, as quantified by micro-computed tomography (µCT). By contrast, PTX increased only bone resorption in T cell knockout (KO) mice, causing net bone loss. Reconstituting T cell-deficient mice with WT, but not Wnt-10b knockout (KO) CD8+ T cells, rescued bone formation and prevented bone loss. To study the role of cAMP signaling in Wnt-10b expression, reverse-transcription polymerase chain reaction (RT-PCR) and luciferase-reporter assays were performed using primary T cells. PDE inhibitors intensified Wnt-10b promoter activity and messenger RNA (mRNA) accumulation in CD3 and CD28 activated CD8+ T cells. In contrast, inhibiting the cAMP pathway mediators protein kinase A (PKA) and cAMP response element-binding protein (CREB), suppressed Wnt-10b expression by T cells activated in the absence of CD28 co-stimulation. In conclusion, the data demonstrate a key role for Wnt-10b production by CD8+ T cells in the bone anabolic response to PDE-inhibitors and reveal competing T cell-independent pro-resorptive properties of PTX, which dominate under T cell-deficient conditions. Selective targeting of CD8+ T cells by PDE inhibitors may be a beneficial approach for promoting bone regeneration in osteoporotic conditions. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

ThPOK Inhibits Osteoclast Formation Via NFATc1 Transcription and Function.

Both LRF (Zbtb7a) and ThPOK (Zbtb7b) belong to the POK (BTB/POZ and Kruppel) family of transcription repressors that participate in development, differentiation, and oncogenesis. Although LRF mediates osteoclast differentiation by regulating NFATc1 expression, the principal established function of ThPOK is transcriptional control of T-cell lineage commitment. Whether ThPOK affects osteoclast formation or function is not known. We find that marrow macrophage ThPOK expression diminishes with exposure to receptor activator of NF-kB ligand (RANKL), but ThPOK deficiency does not affect osteoclast differentiation. On the other hand, enhanced ThPOK, in macrophages, substantially impairs osteoclastogenesis. Excess ThPOK binds the NFATc1 promoter and suppresses its transcription, suggesting a mechanism for its osteoclast inhibitory effect. Despite suppression of osteoclastogenesis by excess ThPOK being associated with diminished NFATc1, osteoclast formation is not rescued by NFATc1 overexpression. Thus, ThPOK appears to inhibit NFATc1 transcription and its osteoclastogenic capacity, while its depletion has no effect on the bone-resorptive cell. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Dmp1 Lineage Cells Contribute Significantly to Periosteal Lamellar Bone Formation Induced by Mechanical Loading But Are Depleted from the Bone Surface During Rapid Bone Formation.

Previous work has shown that osteoprogenitor cells (Prx1+) and pre-osteoblasts (Osx+) contribute to mechanical loading-induced bone formation. However, the role of mature Dmp1-expressing osteoblasts has not been reported. In this study we assessed the contribution of osteoblast lineage cells to bone formation at an early time point following mechanical loading (day 8 from onset of loading). We labeled Osx-expressing and Dmp1-expressing cells in inducible Osx and Dmp1 reporter mice (iOsx-Ai9, iDmp1-Ai9), respectively, 3 weeks before loading. Mice were then loaded daily for 5 days (days 1-5) and were dosed with 5-ethynyl-2'-deoxyuridine (EdU) in their drinking water until euthanasia on day 8. Mice were loaded to lamellar and woven bone inducing stimulation (-7 N/1400 µÎµ, -10 N/2000 µÎµ) to assess differences in these processes. We found varied responses in males and females to the loading stimuli, inducing modest lamellar (females, -7 N), moderate lamellar (males, -10 N), and robust woven (females, -10 N) bone. Overall, we found that preexisting (ie, lineage positive) Osx-expressing and Dmp1-expressing cells contribute largely to the bone formation response, especially during modest bone formation, while our results stuggest that other (non-lineage-positive) cells support the sustained bone formation response during rapid bone formation. With moderate or robust levels of bone formation, a decrease in preexisting Osx-expressing and Dmp1-expressing cells at the bone surface occurred, with a near depletion of Dmp1-expressing cells from the surface in female mice loaded to -10 N (from 52% to 11%). These cells appeared to be replaced by lineage-negative cells from the periosteum. We also found a dose response in proliferation, with 17% to 18% of bone surface cells arising via proliferation in modest lamellar, 38% to 53% in moderate lamellar, and 59% to 81% in robust woven bone formation. In summary, our results show predominant contributions by preexisting Osx and Dmp1 lineage cells to loading-induced lamellar bone formation, whereas recruitment of earlier osteoprogenitors and increased cell proliferation support robust woven bone formation. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

A Subset of Osteosarcoma Bears Markers of CXCL12-Abundant Reticular Cells.

Currently, the cell of origin for osteosarcoma or other primary skeletal tumors is largely unknown. Recent reports identifying specific cell types comprising bone now newly enable investigation of this topic. Specifically, CXC motif chemokine 12 (CXCL12)-abundant reticular (CAR) cells are a specific skeletal stromal cell type that orchestrate the bone marrow microenvironment through cross-talk with hematopoietic and endothelial cells and a likely candidate cell of origin for at least a subset of primary skeletal tumors. Here, we analyze osteosarcomas via immunohistochemistry for known markers of CAR cells such as leptin receptor (LEPR), B-cell factor 3 (EBF3), CXCL12, and platelet-derived growth factor receptor alpha (PDGFRA). A large proportion of high-grade tumors expressed LEPR, PDGFRA, and EBF3 but not CXCL12. These data raise the hypothesis that CAR cells are the cell of origin of this osteoblastic osteosarcoma subset, a finding with implications for the cellular oncogenesis of primary osteosarcoma and the development of effective targeted therapies. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Integrated View on the Role of Vitamin D Actions on Bone and Growth Plate Homeostasis.

1,25(OH)2D3, the biologically active form of vitamin D3, is a major regulator of mineral and bone homeostasis and exerts its actions through binding to the vitamin D receptor (VDR), a ligand-activated transcription factor that can directly modulate gene expression in vitamin D-target tissues such as the intestine, kidney, and bone. Inactivating VDR mutations or vitamin D deficiency during development results in rickets, hypocalcemia, secondary hyperparathyroidism, and hypophosphatemia, pointing to the critical role of 1,25(OH)2D3-induced signaling in the maintenance of mineral homeostasis and skeletal health. 1,25(OH)2D3 is a potent stimulator of VDR-mediated intestinal calcium absorption, thus increasing the availability of calcium required for proper bone mineralization. However, when intestinal calcium absorption is impaired, renal calcium reabsorption is increased and calcium is mobilized from the bone to preserve normocalcemia. Multiple cell types within bone express the VDR, thereby allowing 1,25(OH)2D3 to directly affect bone homeostasis. In this review, we will discuss different transgenic mouse models with either Vdr deletion or overexpression in chondrocytes, osteoblasts, osteocytes, or osteoclasts to delineate the direct effects of 1,25(OH)2D3 on bone homeostasis. We will address the bone cell type-specific effects of 1,25(OH)2D3 in conditions of a positive calcium balance, where the amount of (re)absorbed calcium equals or exceeds fecal and renal calcium losses, as well as during a negative calcium balance, due to selective Vdr knockdown in the intestine or triggered by a low calcium diet. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Higher Levels of Circulating Osteoprogenitor Cells Are Associated With Higher Bone Mineral Density and Lean Mass in Older Adults: A Cross-Sectional Study.

Circulating osteo progenitor (COP) cells are a heterogeneous population of cells that circulate within the peripheral blood with characteristics of the bone marrow mesenchymal stem and progenitor pool. Little is known about the behavior of this cell population in humans. The aim of this study was to identify whether a relationship exists between COP cells (as a percentage of the peripheral blood monocytic cells) and musculoskeletal morphometry and to identify if COP have potential clinical utility as a biomarker for osteoporosis. We recruited 57 older adults (median age: 69 years; IQR: 65, 75 years) living independently in the community and performed cross-sectional analysis to identify associations between the percentage of COP cells and body composition parameters, and through receiver operating characteristic analysis, we evaluated their ability to act as a biomarker of osteoporosis. COP cells were moderately associated with whole-body bone mineral density (BMD) (r = 0.323, p = 0.014) and bone mineral content (BMC) (r = 0.387, p = 0.003), neck of femur BMD (r = 0.473, p < 0.001), and BMC (r = 0.461, p < 0.001) as well as appendicular lean mass (ALM) (p = 0.038) and male sex (p = 0.044) in univariable analysis. In multivariable analysis controlling for age, gender, height, and weight, COP cells remained strongly associated with neck of femur BMD (p = 0.001) and content (p = 0.003). COP cells were also a good predictor of osteoporosis (dual-energy X-ray absorptiometry [DXA] T-score < -2.5) at the neck of femur (cutoff: 0.4%; sensitivity: 100%; specificity 79%) and total body (cutoff: 0.35%; sensitivity: 80%; specificity: 81%). This study shows strong relationships between bone parameters and COP cell number and male sex. They also have potential as a biomarker of osteoporosis, which may provide a new tool for advanced detection and screening in clinical settings. Future larger evaluation studies should verify the cutoffs for biomarker use, and further explore the relationship between COP cells and muscle. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.