Journal of Bone and Mineral Research

In the United States, there are over ten million adults diagnosed with osteoporosis and many more are at risk of developing the condition. Osteoporosis affects both males and females, mostly post-menopausal. Bisphosphonates and denosumab have been widely used globally to treat the condition. The use of bisphosphonates and denosumab had been associated with rare adverse effects including osteonecrosis of the jaw, ONJ, and atypical femur fracture, AFF. However, it remained unclear whether those side effects were class-wide or drug-specific. By analyzing over 230,000 osteoporosis patient reports from the FDA adverse event reporting system, FAERS, we confirmed the association of bisphosphonates and denosumab use with AFF and ONJ. Additionally, comparing each of the four frequently used bisphosphonates with denosumab-treated patients used as a control, we identified: (i) varying significance of association with ONJ and AFF for alendronate, risedronate, ibandronate and zoledronic acid, (ii) over two fold increase in risk of both side effects in alendronate patients, particularly in females, (iii) over a six fold increase in AFF risk in both males and females taking risedronate, and (iv) lower risk of both AFF and ONJ, for zoledronic acid patients compared to denosumab. Key pointsO_LIWe performed a disproportionality analysis of over 230,000 post-marketing reports of patients treated for osteoporosis to measure the risk of developing atypical femur fracture (AFF) and osteonecrosis of the jaw (ONJ). C_LIO_LIAlendronate, ibandronate, risedronate, zoledronic acid, and denosumab were all significantly associated with AFF and ONJ when compared to teriparatide. C_LIO_LIWhen compared to denosumab, patients taking alendronate, ibandronate, risedronate, or zoledronic acid had a variable risk of ONJ and AFF, which correlated with the frequency of drug administration. The trend in variable risk was observed in both females and males. C_LI

Bone fracture is accompanied by mechanical stresses and inflammation - conditions that impair mitochondria via the phenomenon of permeability transition. This phenomenon occurs due to opening of the mitochondrial permeability transition pore (MPTP) promoted by cyclophilin D (CypD). MPTP opening exacerbates inflammation and cell death and, thus can disrupt fracture repair. Here we tested a hypothesis that protecting mitochondria from MPTP opening via inhibition of CypD improves fracture repair. Our data indicate that osteoblast activity, bone formation, and biomechanical properties of repaired bones were significantly increased in CypD knock-out mice when compared to controls during fracture repair. These effects were observed in male but not female mice, thus showing sexual dimorphism. Pharmacological inhibition of CypD with NIM811 in male mice also stimulated fracture repair. In addition, CypD knock-out or pharmacological inhibition produced pro-osteogenic effect in isolated bone marrow osteoprogenitors. This in vitro effect was associated with higher mitochondrial respiration and increased {beta}-catenin activity regulated by mitochondria-dependent acetylation. Our findings implicate a sex-specific role of MPTP in bone fracture and suggest CypD inhibition as a modality to promote fracture repair.

Skull bone mineral density (SK-BMD) provides a suitable trait for the discovery of genes important to bone biology in general, and particularly for identifying components unique to intramembranous ossification, which cannot be captured at other skeletal sites. We assessed genetic determinants of SK-BMD in 43,800 individuals, identifying 59 genome-wide significant loci (4 novel), explaining 12.5% of its variance. Pathway and enrichment analyses of the association signals resulted in clustering within gene-sets involved in regulating the development of the skeleton; overexpressed in the musculoskeletal system; and enriched in enhancer and transcribed regions in osteoblasts. From the four novel loci (mapping to ZIC1, PRKAR1A, ATP6V1C1, GLRX3), two (ZIC1 and PRKAR1A) have previously been related to craniofacial developmental defects. Functional validation of skull development in zebrafish revealed abnormal cranial bone initiation that culminated in ectopic sutures and reduced BMD in mutated zic1 and atp6v1c1 fish and asymmetric bone growth and elevated BMD in mutated prkar1a fish. We confirmed a role of ZIC1 loss-of-function in suture patterning and discovered ATP6V1C1 gene associated with suture development. In light of the evidence presented suggesting that SK-BMD is genetically related to craniofacial abnormalities, our study opens new avenues to the understanding of the pathophysiology of craniofacial defects and towards the effective pharmacological treatment of bone diseases.

Human genetic studies have nominated Cadherin-like and PC-esterase Domain-containing 1 (CPED1) as a candidate target gene mediating bone mineral density (BMD) and fracture risk heritability. Recent efforts to define the role of CPED1 in bone in mouse and human models have revealed complex alternative splicing and inconsistent results arising from gene targeting, making its function in bone difficult to interpret. To better understand the role of CPED1 in adult bone mass and morphology, we conducted a comprehensive genetic and phenotypic analysis of cped1 in zebrafish, an emerging model for bone and mineral research. We analyzed two different cped1 mutant lines and performed deep phenotyping to characterize more than 200 measures of adult vertebral, craniofacial, and lean tissue morphology. We also examined alternative splicing of zebrafish cped1 and gene expression in various cell/tissue types. Our studies fail to support an essential role of cped1 in adult zebrafish bone. Specifically, homozygous mutants for both cped1 mutant alleles, which are expected to result in loss-of-function and impact all cped1 isoforms, exhibited no significant differences in the measures examined when compared to their respective wildtype controls, suggesting that cped1 does not significantly contribute to these traits. We identified sequence differences in critical residues of the catalytic triad between the zebrafish and mouse orthologs of CPED1, suggesting that differences in key residues, as well as distinct alternative splicing, could underlie different functions of CPED1 orthologs in the two species. Our studies fail to support a requirement of cped1 in zebrafish bone and lean tissue, adding to evidence that variants at 7q31.31 can act independently of CPED1 to influence BMD and fracture risk. Lay summaryBone mineral density (BMD) is a key indicator for predicting and diagnosing osteoporosis and fracture risk, and it has been estimated that up to 89% of variation in BMD is determined by genetics. Multiple human genetics studies have nominated CPED1 as a potential gene underlying BMD and fracture risk heritability, however the function of CPED1 remains poorly understood. In this study, we examined the role of cped1 in bone by quantifying over 200 morphological measures of vertebral and craniofacial bone size, shape, and density in two different mutant lines of zebrafish in which cped1 function was reduced or eliminated. We also examined lean tissue mass because co-heritability of this trait with BMD has also been hypothesized to involve CPED1. Surprisingly, despite the loss of cped1 function, there were no significant differences between the mutant zebrafish and their respective controls. Our study therefore fails to support a role for cped1 in bone and lean tissue, suggesting that hereditary influence on BMD and fracture risk can occur independently of CPED1.

RNF43 and ZNRF3 are transmembrane E3 ubiquitin ligases that negatively regulate Wnt signaling by promoting ubiquitination and degradation of Frizzled receptors. Loss of either gene enhances Wnt/{beta}-catenin signaling and has been linked to tumorigenesis. Wnt signaling is a key regulator of skeletal development and bone homeostasis, and pharmacologic activation of this pathway is an established therapy for osteoporosis. In Xenopus laevis, simultaneous disruption of rnf43 and znrf3 results in supernumerary limb formation; however, their roles in mammalian limb development and skeletal maintenance remain unclear. We demonstrate that mice homozygous for null alleles of both Rnf43 and Znrf3 do not develop supernumerary limbs. Because activation of Wnt/{beta}-catenin signaling in osteoblasts increases bone mass, we hypothesized that osteoblast-specific deletion of Rnf43 and/or Znrf3 would produce a high-bone-mass phenotype. Instead, osteoblast-specific loss of Znrf3 resulted in age-and sex-dependent reductions in trabecular bone mass, characterized by decreased bone mineral density and bone volume fraction, reduced trabecular number, and increased trabecular separation. Cortical bone exhibited increased cross-sectional size with reduced cortical area fraction and altered structural properties, while tissue mineral density was unchanged. In contrast, deletion of Rnf43 had minimal skeletal effects, and combined deletion of both genes did not exacerbate the phenotype observed with loss of Znrf3 alone. These findings identify Znrf3 as the dominant functional paralog regulating bone architecture in mature osteoblasts and underscore the importance of evaluating skeletal geometry when modulating upstream Wnt regulators.

Fibrous dysplasia (FD) is a rare disorder associated with fractures and deformities. FD lesions produce excess phosphaturic hormone fibroblast growth factor 23 (FGF23), leading to hyperphosphaturia in most patients, and hypophosphatemia in those with high FD burden. Skeletal complications are associated with both low-normophosphatemia and frank hypophosphatemia. Burosumab is approved for other forms of FGF23 excess, but there is little evidence to inform use in FD. A phase 2 study investigated the safety and efficacy of burosumab in patients with FD. The primary endpoint was the proportion of participants achieving phosphate levels within a high-normal target range (age and sex-adjusted Z-score -1 to +2). 12 participants (7 children, 5 adults) received burosumab for 48 weeks. Median phosphate Z-score increased from -2.88 (1.65) to 0.22 (1.37), meeting the target in 100% of participants. Alkaline phosphatase levels were elevated at baseline in 8 participants and declined by 49%. PROMIS questionnaires showed trends toward improvements in all domains in children; adult scores showed no identifiable trends. Two children experienced transformational mobility gains, including advancement from full-time wheelchair use to independent ambulation. Lesion biopsies showed no changes in cellularity or composition, and 18F-NaF PET/CT scans showed no changes in tracer uptake, suggesting burosumab did not adversely impact lesional activity. Adverse events were mild, and none resulted in treatment withdrawal. Burosumab targeting high-normophosphatemia in patients with FD was well-tolerated, restored phosphate homeostasis, and improved bone turnover. Burosumab has the potential to lead to functional improvements and ambulation gains in severely affected patients and is a valuable tool to reduce the impact of FD-related disability.

Fracture Liaison Services (FLSs) are recommended healthcare models to deliver secondary fracture prevention and reduce the risk of subsequent fractures. Several studies have demonstrated the cost and clinical effectiveness of FLSs, but there is little real-world data on the impact of FLSs on subsequent hip fracture rates. A cohort of 50,214 patients from the national FLS database with an index fracture of the hip, spine, or other in 2017 was linked to the National Hip Fracture Database from 2017 to 2020 to identify those patients who went on to have a subsequent hip fracture. One in twenty (5.1%) of the 9,888 people in whom the index fracture was at the hip went on to suffer a second hip fracture within 3-4 years, despite receiving the support of an FLS. The risk of hip fracture was similar (4.7%) if the index fracture was at the spine, but lower at other sites (2.8%, p<0.001) and the interval shortest after an index hip fracture (1.1 years (0.4,2.0) p<0.001). The proportion of patients with a subsequent hip fracture was not lower by types of anti-osteoporotic medication. This work highlights the need for alternative anti-osteoporotic management strategies to rapidly decrease the risk of subsequent hip fractures for people seen by an FLS setting with levels of risk that are even higher for patients in areas which are still not served by an FLS.

Chondrocytes within the fracture callus transform into osteoblasts during bone regeneration, but the molecular mechanisms regulating this process are unknown. Wnt ligands are expressed within the fracture callus, and hypertrophic chondrocytes undergoing transformation to osteoblasts exhibit nuclear localization of {beta}-catenin, indicating active Wnt signaling in these cells. Here, we show that conditional knock out (cKO) of {beta}-catenin in chondrocytes inhibits the transformation of chondrocytes to osteoblasts, while stabilization of {beta}-catenin in chondrocytes accelerates this process. After cKO, chondrocyte-derived cells were located in the bone marrow cavity and upon re-fracture formed cartilage. Lineage tracing in wild type mice revealed that in addition to osteoblasts, chondrocytes give rise to stem cells that contribute to repair of subsequent fractures. These data indicate that Wnt signaling directs cell fate choices of chondrocytes during fracture healing by stimulating transformation of chondrocytes to osteoblasts, and provide a framework for developing Wnt-therapies to stimulate repair.

We conducted genome-wide association studies (GWAS) of dual-energy X-ray absorptiometry (DXA)-derived bone mineral density (BMD) traits at 11 skeletal sites, within over 30,000 European individuals from the UK Biobank. A total of 92 unique and independent loci were identified for 11 DXA-derived BMD traits and fracture, including five novel loci (harboring genes such as ABCA1, CHSY1, CYP24A1, SWAP70, and PAX1) for six BMD traits. These loci exhibited evidence of association in both males and females, which could serve as independent replication. We demonstrated that polygenic risk scores (PRSs) were independently associated with fracture risk. Although incorporating multiple PRSs (metaPRS) with the clinical risk factors (i.e., the FRAX model) exhibited the highest predictive performance, the improvement was marginal in fracture prediction. The metaPRS were capable of stratifying individuals into different trajectories of fracture risk, but clinical risk factors played a more significant role in the stratification. Additionally, we uncovered genetic correlation and shared polygenicity between head BMD and intracranial aneurysm. Finally, by integrating gene expression and GWAS datasets, we prioritized genes (e.g. ESR1 and SREBF1) encoding druggable human proteins along with their respective inhibitors/antagonists. In conclusion, this comprehensive investigation revealed a new genetic basis for BMD and its clinical relevance on fracture prediction. More importantly, it was suggested that head BMD was genetically correlated with intracranial aneurysm. The prioritization of genetically supported targets implied the potential repurposing drugs (e.g. the n-3 PUFA supplement targeting SREBF1) for the prevention of osteoporosis.

To optimize osteoporosis therapy with the parathyroid hormone fragment teriparatide (PTH1-34), we sought to determine whether strontium (Sr) could potentiate the bone anabolic action of intermittent PTH1-34 in ovariectomized rats. Female rats were either Sham-operated or ovariectomized (Ovx) at 6 months of age. Eight weeks after surgery, Ovx rats received either vehicle solutions, 625 mg/kg/day Sr (5 days per week), 8 {micro}g/kg/day PTH1-34 (5 days per week), or the combined treatments for 8 weeks. PTH1-34 reversed Ovx-induced deterioration of trabecular microarchitecture, apparent volumetric bone mineral density (vBMD), and strength, whereas Sr alone increased tissue-level vBMD without significantly affecting trabecular bone mass. Co-treatment with Sr and PTH1-34 further increased trabecular thickness, apparent and tissue-level vBMD, bone material properties (force and working energy), and trabecular bone strength compared with PTH1-34 alone. In cortical bone, PTH1-34 increased bone volume, cortical thickness, and apparent vBMD, while co-treatment further enhanced cortical thickness and apparent vBMD, maintained the Sr-induced increase in tissue-level vBMD, and significantly improved bone strength. In primary osteoblast cultures, Sr and PTH1-34, administered either alone or in combination, increased Rankl and decreased Opg expression, consistent with the elevated urinary levels of the bone resorption marker deoxypyridinoline in vivo. Sr or PTH1-34 alone stimulated Igf1 and Alpl expression, whereas co-stimulation further enhanced these osteogenic markers. In conclusion, combining Sr with PTH1-34 integrates the osteoanabolic effects of PTH1-34 on bone mass with the mineral-level effects of Sr on bone material properties, leading to synergistic stimulation of bone formation and superior improvements in bone quality and strength.

Fractures heal by rapid formation of mineralized callus. Essential to this process is proliferation of periosteal cells to supply bone-forming osteoblasts. To better understand the role of cell proliferation in fracture healing, we asked: How is callus composition altered when osteolineage cells proliferation is blocked? Do mature osteoblasts proliferate to contribute to callus formation? First, mice expressing herpes simplex virus-thymidine kinase (HSV-TK) driven by the 3.6Col1a1 promoter were treated with ganciclovir (GCV) to ablate proliferating osteolineage cells for 5 or 10 days. Analysis of callus cells using single-cell RNA-seq revealed that GCV-treated Col1-TK mice had fewer osteoblasts and chondrocytes than control mice, with more myofibroblasts and immune cells, consistent with fibrous nonunion. In controls, 15-30% of callus cells that expressed the early osteoblast markers osterix (OSX) and the late marker osteocalcin (OCN) were in the cell cycle. Next, we targeted proliferating osteoblasts at different stages of differentiation by crossing Osx-CreERT2, Ocn-Cre and Dmp1-CreERT2 mice with novel ROSA-TK mice. Following fracture, each Cre;ROSA-TK mouse line exhibited poorer radiographic healing, decreased callus bone volume and a shift from callus bone to fibrous tissue. We conclude that osteoblasts, often considered post-mitotic, proliferate after fracture to contribute to formation of mineralized callus essential to healing.

Heterotopic ossification (HO), either acquired or hereditary, is featured by ectopic bone formation outside of the normal skeleton. The acquired form of HO is a debilitating and common complication of musculoskeletal trauma, central nervous system injury, burns, combat trauma, hip and elbow fractures, and total joint replacement surgeries. It can be characterized as abnormal bone formation that occurs mostly by endochondral ossification. Recent studies have implicated inflammation and dysregulation of Hedgehog (Hh) signaling as major early contributors to HO formation. Here, we demonstrate that administration of the Hh pathway inhibitor, arsenic trioxide (ATO), prevented acquired HO in a clinically-relevant trauma/burn mouse model. We further evaluated the effects of two additional Hh pathway antagonists: cholecalciferol and pravastatin on mitigating osteoblast differentiation. Finally, we assessed the effect of a combination of Hh pathway inhibitors on reducing systemic proinflammatory responses. A targeted combination approach using Hh pathway inhibitors may offer potential therapeutic benefits though targeting differential components of the Hh pathway. Taken together, our study demonstrates that the administration of single or multiple Hh pathway inhibitors may have the potential to reduce the formation of acquired HO.

BACKGROUNDFibrous dysplasia (FD) is a rare, disabling disease with no established treatments. Growing evidence supports inhibiting the pro-osteoclastic factor receptor activator of nuclear Kappa-B ligand (RANKL) as a potential treatment strategy. We conducted a phase 2 trial evaluating the anti-RANKL drug denosumab in adults with FD, with an emphasis on investigating post-discontinuation bone turnover rebound, and cellular mechanisms underlying anti-RANKL effects on FD osteoprogenitors. METHODSEight subjects received denosumab for 6-months and were observed for 8-months post-discontinuation. Efficacy and safety were evaluated using bone turnover markers, 18F-NaF PET/CT, and lesion biopsies. RANKL neutralization effects were assessed by histology, RNASeq, and an FD mouse model. Interplay between osteoclasts and FD osteoprogenitors was assessed in an ex vivo lesion model. RESULTSDenosumab markedly reduced bone turnover and radiographic lesional activity in all subjects. Denosumab was well-tolerated during the treatment period, however post-discontinuation turnover reached or exceeded pre-treatment in six subjects, associated with severe hypercalcemia in one. Histology and whole-exome RNA sequencing showed reduced FD cell proliferation and increased osteogenic maturation, with increased lesional bone formation. The ex vivo model supported the dependence of FD cell proliferation on osteoclast activation. CONCLUSIONSOsteoclast inhibition by anti-RANKL decreased FD cell proliferation and lesional activity, enabling osteogenic maturation and bone formation. These findings provide new understanding of FD pathogenesis as driven by crosstalk between osteoclasts and pre-osteoblast/osteoblasts, and support denosumab as a mechanistically-driven treatment strategy. Marked bone turnover rebound with post-discontinuation hypercalcemia occurs in a subset of patients, particularly younger individuals with high disease burden. TRIAL REGISTRATIONClinicalTrials.gov NCT03571191 FUNDINGThis work was supported by the Intramural Research Program of the NIDCR, NICHD, and Clinical Center, National Institutes of Health. Clinical trial NCT03571191 was conducted as an investigator-sponsored study supported by Amgen, Inc. This research was supported in part by the NIDCR Genomics and Computational Biology Core: ZIC DC000086 and Veterinary Resources Core: ZIC DE000740-05. Work in MTC lab and LVC labs were supported by the of Research on Womens Health (ORWH) through the Bench to Bedside Program award #884515.

Emerging evidence supports that osteogenic differentiation of skeletal stem cells (SSCs) is a key determinant of overall bone formation and bone mass. Despite extensive studies showing mitogen-activated protein kinase (MAPK) function in osteoblast differentiation, none of these studies properly show in vivo evidence of impacting post-lineage commitment and subsequent maturation. Here, we describe how the extracellular signal-regulated kinase (ERK) pathway in osteoblasts controls bone formation by suppressing the mechanistic target of rapamycin (mTOR) pathway. We also show that, while ERK inhibition blocks the differentiation of osteogenic precursors when initiated at an early stage, ERK inhibition surprisingly promotes the later stages of osteoblast differentiation. Accordingly, inhibition of the ERK pathway using a small compound inhibitor or conditional deletion of the MAP2Ks Mek1 and Mek2, in mature osteoblasts and osteocytes (Mek1/2Dmp1), markedly increased bone formation due to augmented osteoblast differentiation. Mice with inducible deletion of the ERK pathway in mature osteoblasts (Mek1/2Ocn-Ert) also displayed similar phenotypes, demonstrating that this phenotype reflects continuous postnatal inhibition of late-stage osteoblast maturation. Mechanistically, ERK inhibition increases mitochondrial function and SGK1 phosphorylation via mTOR2 activation, which leads to osteoblast differentiation and production of angiogenic and osteogenic factors to promote bone formation. This phenotype was partly reversed by inhibiting mTOR. Our study uncovers a surprising dichotomy of ERK pathway functions in osteoblasts, whereby ERK activation promotes the early differentiation of osteoblast precursors, but inhibits the subsequent differentiation of committed osteoblasts via mTOR-mediated regulation of mitochondrial function and SGK1.

In adult mice, new bone accrual following mechanical load is mediated by the neurotrophin nerve growth factor (NGF) that is expressed by osteoblasts on the bone surface. NGF can bind to its high affinity receptor, neurotrophic tyrosine kinase receptor type 1 (TrkA), on peripheral sensory nerves resident in bone and support new bone formation. However, the osteoanabolic therapeutic potential of NGF-TrkA signaling to repair bone is limited due to the long-lasting thermal and mechanical hyperalgesia induced by administration of NGF in mice and humans. Here, we investigated whether 1) mature osteoblasts are the primary source of NGF required for bone accrual following loading, and 2) a small molecule TrkA receptor agonist - gambogic amide - can harness the downstream osteoanabolic potential of NGF-TrkA signaling in the absence of endogenous NGF. Loss of Ngf transcription in mature osteoblasts did not appear to affect bone structure or bone mass in adulthood. However, Ngf knockout mice significantly reduced periosteal bone accrual and osteogenic Wnt transcription in response to loading compared to wildtype mice. Intraperitoneal injection of gambogic amide prior to loading was unable to produce its osteoanabolic effects in Ngf knockout mice, suggesting that gambogic amide primarily functions in collaboration with endogenous NGF in bone. In total, our study reveals an important role for osteoblastic NGF in the skeletal adaptation of bone to mechanical forces.

Osteoporosis is a prevalent cause of fractures in older adults and remains a source of morbidity that requires efforts to develop therapeutics. Circulating proteins play a critical role in the pathophysiology of osteoporosis and offer opportunities to identify new causal determinants of bone health. We therefore performed a large-scale proteome-wide Mendelian randomization (MR) analysis to estimate the effects of genetically determined circulating proteins levels on bone mineral density (BMD) and fracture risk. Genetic instruments were derived from cis-protein quantitative trait loci (cis-pQTLs) for 2,110 plasma proteins across four European ancestry cohorts and applied to genome-wide association studies (GWAS) of heel estimated BMD, femoral neck BMD, lumbar spine BMD, any fracture, and forearm fracture in up to 426,824 individuals of European ancestry. Across proteins and outcomes, 192 protein-skeletal outcome associations showed MR evidence of association, without evidence for heterogeneity or horizontal pleiotropy, and 128 of these further showed strong colocalization with osteoporosis-related loci. We then prioritized proteins that replicated across cohorts, exhibited concordant effect directions, and were likely to be active in circulation, yielding 18 high-confidence causal proteins for BMD and fracture risk. These included established skeletal regulators such as sclerostin (SOST) and R-spondin-3 (RSPO3), which showed opposing effects consistent with their known biology, along with less well-characterized proteins. Higher genetically predicted tissue inhibitor of metalloproteinases 2 (TIMP2) levels was associated with lower BMD and increased forearm fracture risk. Gene-level and variant-level phenome-wide association analyses converged on skeletal traits, and rare predicted damaging or loss-of-function variants in TIMP2 were associated with higher BMD at the heel, spine and hip. Our findings implicate several circulating proteins as putatively causal factors for osteoporosis and, among them, provide multiple layers of evidence supporting TIMP2 as a genetically supported candidate for further functional and translational evaluation. Lay summaryOsteoporosis is characterized by decreased bone density, and despite available medications, it remains a key risk factor for fractures, requiring continued effort for development of new therapeutics. We used genetic data to estimate the effect of genetically predicted levels of 2,110 blood proteins on strength and fracture risk. We found 18 proteins with effects on bone mineral density and fractures. One protein, tissue inhibitor of metalloproteinases 2 (TIMP2), showed robust evidence linking its higher levels to decreased bone density and increased risk of forearm fracture, highlighting TIMP2 as a promising new treatment target for osteoporosis.

Autophagy is a recycling pathway in which damaged or dysfunctional proteins, protein aggregates, and organelles are delivered to lysosomes for degradation. Insufficiency of autophagy is thought to contribute to several age-related diseases including osteoporosis. Consistent with this, elimination of autophagy from the osteoblast lineage reduces bone formation and causes low bone mass. However, whether increasing autophagy would benefit bone health is unknown. Here, we increased expression of the endogenous Transcription Factor EB gene (Tfeb) in osteoblast lineage cells in vivo via CRISPR activation. Tfeb overexpression stimulated autophagy and lysosomal biogenesis in osteoblasts. Tfeb overexpressing male mice displayed a robust increase in femoral and vertebral cortical thickness at 4.5 months of age. Histomorphometric analysis revealed that the increase in femoral cortical thickness was due to increased bone formation at the periosteal surface. Tfeb overexpression also increased femoral trabecular bone volume. Consistent with these results, bone strength was increased in Tfeb overexpressing mice. Female Tfeb overexpressing mice also displayed a progressive increase in bone mass over time and at 12 months of age had high cortical thickness and trabecular bone volume. This increase in vertebral trabecular bone volume was due to elevated bone formation. Osteoblastic cultures showed that Tfeb overexpression increased proliferation and osteoblast formation. Overall, these results demonstrate that stimulation of autophagy in osteoblast lineage cells promotes bone formation and strength and may represent an effective approach to combat osteoporosis.

BACKGROUNDSOsteopetrosis, a rare skeletal disease, is characterized by an increased bone mass resulting from impaired bone remodeling process. Platelet is the major bone-healing blood component involved in the regulation of bone resorption, particularly in the removal of compromised bones. Several actin-associated proteins contribute to the orchestration of actin ring formation in osteoclasts closely related to bone resorption. However, the role of coactosin-like F-actin binding protein 1 (Cotl1) in actin ring formation and platelet-mediated bone resorption mechanisms remains unclear. METHODSWhole-mount in situ RNA hybridization was performed to detect cotl1 expression pattern in zebrafish. cotl1 gene knockdown zebrafish using morpholino oligonucleotides and platelet marker-expressing transgenic zebrafish were investigated for finding the phenotypic clues. Cotl1 knockout (Cotl1-/-) mice were generated using Cre/loxP recombination systems. In silico network analysis of the differentially expressed genes between bone marrow samples of wild type and Cotl1-/- mice was conducted. Primary-cultured monocytes from Cotl1-/- mice were examined for osteoclast differentiation and mRNA and protein expression patterns. Cotl1-/- mice underwent hematological examination and bone phenotype assessments including micro-CT, bone density, histology, immunohistochemistry, electron microscopy, and mechanical testing. Genetic association of SNPs in human COTL1 gene with estimated bone mineral density was analyzed. RESULTSZebrafish cotl1 mRNA was highly expressed in the caudal hematopoietic tissue region. Knockdown of cotl1 in zebrafish embryos decreased the expression of c-myb, a marker of hematopoietic stem cells (HSCs). Notably, the platelet receptor CD41 was reduced in the HSCs of cotl1-depleted zebrafish and Cotl1-/- mice showed reduced platelet production with platelet surface markers of CD41 and CD61. Significantly reduced osteoclast differentiation and bone resorption pit, and impaired actin ring formation were observed in the primary myocytes from Cotl1-/- mice. Structural and histological analyses of the femur revealed sclerotic bone phenotypes in Cotl1-/- mice. Mechanical assessment of Cotl1-/- mouse femoral bones revealed osteopetrotic phenotypes. Association analysis of genetic variants in COTL1 gene in subjects from the UK Biobank suggested that COTL1 is susceptible to bone density in humans. CONCLUSIONSOur results provide insights into the role of Cotl1 in platelet-mediated osteoclastogenesis and the novel finding that the loss of Cotl1-/- mice causes osteopetrosis phenotypes. Clinical PerspectiveWhat Is New? O_LIDeficiency of Cotl1 decreased platelet production in zebrafish and mice. C_LIO_LIAbsence of Cotl1 disrupted the actin ring formation which is crucial for osteoclast differentiation in bone remodeling process. C_LIO_LICotl1 knockout mice displayed sclerotic bone phenotypes and increased bone density that are representative characteristics of osteopetrosis. C_LIO_LIGenetic variants in COTL1 gene in subjects from the UK Biobank are significantly associated with bone density. C_LI What Are the Clinical Implications? O_LIThe current findings suggest that Cotl1 plays a fundamental role in platelet production-mediated osteoclastogenesis during bone remodeling, providing valuable insights into novel strategies for bone health maintenance. C_LIO_LICotl1 may be a promising target for novel therapeutic strategies for the treatment and/or prevention of impaired osteoclastogenesis-mediated bone diseases such as osteopetrosis and osteoporosis. C_LI

SUMMARYZoledronic acid (ZA) prevents muscle weakness in mice with bone metastases; however, its role in muscle weakness in non-tumor-associated metabolic bone diseases and as an effective treatment modality for the prevention of muscle weakness associated with bone disorders, is unknown. We demonstrate the role of ZA-treatment on bone and muscle using a mouse model of accelerated bone remodeling, which represents the clinical manifestation of non-tumor associated metabolic bone disease. ZA increased bone mass and strength and rescued osteocyte lacunocanalicular organization. Short-term ZA treatment increased muscle mass, whereas prolonged, preventive treatment improved muscle mass and function. In these mice, muscle fiber-type shifted from oxidative to glycolytic and ZA restored normal muscle fiber distribution. By blocking TGF{beta} release from bone, ZA improved muscle function, promoted myoblast differentiation and stabilized Ryanodine Receptor-1 calcium channel. These data demonstrate the beneficial effects of ZA in maintaining bone health and preserving muscle mass and function in a model of metabolic bone disease. Context and significanceTGF{beta} is a bone regulatory molecule which is stored in bone matrix, released during bone remodeling, and must be maintained at an optimal level for the good health of the bone. Excess TGF{beta} causes several bone disorders and skeletal muscle weakness. Reducing excess TGF{beta} release from bone using zoledronic acid in mice not only improved bone volume and strength but also increased muscle mass, and muscle function. Progressive muscle weakness coexists with bone disorders, decreasing quality of life and increasing morbidity and mortality. Currently, there is a critical need for treatments improving muscle mass and function in patients with debilitating weakness. Zoledronic acids benefit extends beyond bone and could also be useful in treating muscle weakness associated with bone disorders.

Myocardial infarction (MI) and osteoporotic fracture (Fx) are leading causes of morbidity and mortality worldwide, and there is epidemiological evidence linking their incidence, suggesting possible crosstalk. MI can exacerbate underlying atherosclerosis through sympathetic nervous system activation and {beta}3 adrenoreceptor-mediated release of hematopoietic stem cells (HSCs), leading to monocytosis. We hypothesized that this same pathway may initiate systemic bone loss following MI, since osteoclasts differentiate from monocytes. In this study, MI was performed in 12-week-old male mice (n=24), and mice were randomized to treatment with a {beta}3-adrenergic receptor antagonist (SR 59230A) or no treatment for 10 days post-operatively; additional mice (n=21, treated and untreated) served as un-operated controls. Bone mineral density (BMD), bone mineral content (BMC), and body composition were quantified at baseline and 10 days post-MI using DXA; at 10 days post-MI circulating monocyte and neutrophil levels were quantified, and the L5 vertebral body and femur were analyzed with micro-computed tomography. We found that MI led to increases in circulating monocyte and neutrophil levels, but contrary to our initial hypothesis, {beta}3-antagonist treatment further increased circulating monocytes compared to untreated mice. BMD and BMC of MI mice decreased at the femur and lumbar spine (-6.9% femur BMD, -3.5% lumbar BMD); {beta}3-antagonist treatment diminished this bone loss response (-5.3% femur BMD, -1.2% lumbar BMD). Similarly, trabecular bone volume was decreased in MI mice compared to control mice, and this bone loss was partially attenuated by {beta}3-antagonist treatment. These results suggest that MI leads to bone loss at both the axial and appendicular skeleton, and that the sympathetic nervous system may be a modulator of this response. This study is the first to mechanistically show bone loss following an ischemic injury; these findings suggest that bone loss and increased fracture risk may be important clinical co-morbidities following MI or other ischemic injuries.