ED-71 Improves Bone Mass in Ovariectomized Rats by Inhibiting Osteoclastogenesis Through EphrinB2-EphB4-RANKL/OPG Axis.

Purpose: Estrogen deficiency is the main reason of postmenopausal osteoporosis. Eldecalcitol (ED-71) is a new active vitamin D analogue clinically used in the treatment of postmenopausal osteoporosis. We aimed to investigate whether EphrinB2-EphB4 and RANKL/RANK/OPG signaling cooperate in mediating the process of osteoporosis by ED-71. Methods: In vivo, the ovariectomized (OVX) rats were administered orally with 30 ng/kg ED-71 once a day for 8 weeks. HE staining, Masson staining and Immunofluorescence staining were used to evaluate bone mass, bone formation, osteoclastogenesis associated factors and the expression of EphrinB2, EphB4, RANKL and OPG. In vitro, H2O2 stimulation was used to simulate the cell environment in osteoporosis. Immunofluorescence, quantitative real time PCR (qRT-PCR), enzyme-linked immunosorbent assay (ELISA) and Western Blot were applied to detect the expression of EphrinB2, EphB4, RANKL and OPG. In osteoblasts, EphB4 was knocked down by EphB4 small-interfering RNA (siRNA) transfection. LY294002 (PI3K inhibitor) or ARQ092 (AKT inhibitor) was used to block PI3K/AKT pathway. An indirect co-culture system of osteoblasts and osteoclasts was established. The mRNA and protein expression of osteoclastogenes is associated factors were tested by qRT-PCR and Western Blot. Results: ED-71 increased bone mass and decreased the number of osteoclasts in OVX rats. Moreover, ED-71 promoted the expression of EphrinB2, EphB4, and decreased the RANKL/OPG ratio in osteoblasts. Osteoclastogenesis was restrained when osteoclasts were indirectly co-cultured with ED-71-treated osteoblasts. After silencing of EphB4 expression in osteoblasts, ED-71 inhibited the expression of P-PI3K and P-AKT and increased the ratio of RANKL/OPG. This reversed the inhibitory effect of ED-71 on osteoclastogenes. Therefore, in ED-71-inhibited osteoclastogenes, EphB4 is a key factor affecting the secretion of RANKL and OPG by osteoblasts. EphB4 suppressed the RANKL/OPG ratio through activating PI3K/AKT signaling in osteoblasts. Conclusion: ED-71 inhibits osteoclastogenesis through EphrinB2-EphB4-RANKL/OPG axis, improving bone mass in ovariectomized rats. PI3K/AKT pathway is involved this process.

Hyperthyroidism-driven bone loss depends on BMP receptor Bmpr1a expression in osteoblasts.

Hyperthyroidism is a well-known trigger of high bone turnover that can lead to the development of secondary osteoporosis. Previously, we have shown that blocking bone morphogenetic protein (BMP) signaling systemically with BMPR1A-Fc can prevent bone loss in hyperthyroid mice. To distinguish between bone cell type-specific effects, conditional knockout mice lacking Bmpr1a in either osteoclast precursors (LysM-Cre) or osteoprogenitors (Osx-Cre) were rendered hyperthyroid and their bone microarchitecture, strength and turnover were analyzed. While hyperthyroidism in osteoclast precursor-specific Bmpr1a knockout mice accelerated bone resorption leading to bone loss just as in wildtype mice, osteoprogenitor-specific Bmpr1a deletion prevented an increase of bone resorption and thus osteoporosis with hyperthyroidism. In vitro, wildtype but not Bmpr1a-deficient osteoblasts responded to thyroid hormone (TH) treatment with increased differentiation and activity. Furthermore, we found an elevated Rankl/Opg ratio with TH excess in osteoblasts and bone tissue from wildtype mice, but not in Bmpr1a knockouts. In line, expression of osteoclast marker genes increased when osteoclasts were treated with supernatants from TH-stimulated wildtype osteoblasts, in contrast to Bmpr1a-deficient cells. In conclusion, we identified the osteoblastic BMP receptor BMPR1A as a main driver of osteoporosis in hyperthyroid mice promoting TH-induced osteoblast activity and potentially its coupling to high osteoclastic resorption.

mPPTMP195 nanoparticles enhance fracture recovery through HDAC4 nuclear translocation inhibition.

Delayed repair of fractures seriously impacts patients' health and significantly increases financial burdens. Consequently, there is a growing clinical demand for effective fracture treatment. While current materials used for fracture repair have partially addressed bone integrity issues, they still possess limitations. These challenges include issues associated with autologous material donor sites, intricate preparation procedures for artificial biomaterials, suboptimal biocompatibility, and extended degradation cycles, all of which are detrimental to bone regeneration. Hence, there is an urgent need to design a novel material with a straightforward preparation method that can substantially enhance bone regeneration. In this context, we developed a novel nanoparticle, mPPTMP195, to enhance the bioavailability of TMP195 for fracture treatment. Our results demonstrate that mPPTMP195 effectively promotes the differentiation of bone marrow mesenchymal stem cells into osteoblasts while inhibiting the differentiation of bone marrow mononuclear macrophages into osteoclasts. Moreover, in a mouse femur fracture model, mPPTMP195 nanoparticles exhibited superior therapeutic effects compared to free TMP195. Ultimately, our study highlights that mPPTMP195 accelerates fracture repair by preventing HDAC4 translocation from the cytoplasm to the nucleus, thereby activating the NRF2/HO-1 signaling pathway. In conclusion, our study not only proposes a new strategy for fracture treatment but also provides an efficient nano-delivery system for the widespread application of TMP195 in various other diseases.

Hypoxic dental pulp stem cells-released succinate promotes osteoclastogenesis and root resorption.

Introduction: Clinical studies have shown that endodontically-treated nonvital teeth exhibit less root resorption during orthodontic tooth movement. The purpose of this study was to explore whether hypoxic dental pulp stem cells (DPSCs) can promote osteoclastogenesis in orthodontically induced inflammatory root resorption (OIIRR). Methods: Succinate in the supernatant of DPSCs under normal and hypoxic conditions was measured by a succinic acid assay kit. The culture supernatant of hypoxia-treated DPSCs was used as conditioned medium (Hypo-CM). Bone marrow-derived macrophages (BMDMs) from succinate receptor 1 (SUCNR1)-knockout or wild-type mice were cultured with conditioned medium (CM), exogenous succinate or a specific inhibitor of SUCNR1 (4c). Tartrate-resistant acid phosphatase (TRAP) staining, Transwell assays, qPCR, Western blotting, and resorption assays were used to evaluate osteoclastogenesis-related changes. Results: The concentration of succinate reached a maximal concentration at 6 h in the supernatant of hypoxia-treated DPSCs. Hypo-CM-treated macrophages were polarized to M1 proinflammatory macrophages. Hypo-CM treatment significantly increased the formation and differentiation of osteoclasts and increased the expression of osteoclastogenesis-related genes, and this effect was inhibited by the specific succinate inhibitor 4c. Succinate promoted chemotaxis and polarization of M1-type macrophages with increased expression of osteoclast generation-related genes. SUCNR1 knockout decreased macrophage migration, M1 macrophage polarization, differentiation and maturation of osteoclasts, as shown by TRAP and NFATc1 expression and cementum resorption. Conclusions: Hypoxic DPSC-derived succinate may promote osteoclast differentiation and root resorption. The regulation of the succinate-SUCNR1 axis may contribute to the reduction in the OIIRR.

mmu-miR-185 regulates osteoclasts differentiation and migration by targeting Btk.

BACKGROUND: Bones undergo a constant remodeling, a process involving osteoclast-mediated bone resorption and osteoblast-mediated bone formation, crucial for maintaining healthy bone mass. We previously observed that miR-185 depletion may promote bone formation by regulating Bgn expression and the BMP/Smad signaling pathway. However, the effects of miR-185-5p on the osteoclasts and bone remodeling have not been elucidated, warranting further exploration. METHODS: Tartrate-resistant acid phosphatase staining was utilized to assess the differentiation ability of bone marrow mononuclear macrophages (BMMs) from mmu-miR-185 gene knockout (KO) mice and wild-type (WT) mice. A reverse transcriptase-quantitative PCR was conducted to compare differences in miR-185-5p and osteoclast marker molecules, including Trap, Dcstamp, Ctsk and Nfatc1, between the KO group and WT group BMMs. Western blot analysis was employed to observe the expression of osteoclast marker molecules. A cell-counting kit-8 was used to analyze cell proliferation ability. Transwell experiments were conducted to detect cell migration. Dual-luciferase reporter assays were employed to confirm whether Btk is a downstream target gene of miR-185-5p. RESULTS: miR-185 depletion promoted osteoclast differentiation in bone marrow-derived monocytes/macrophages. Overexpression of miR-185-5p in RAW264.7 cells inhibited differentiation and migration of osteoclasts. Furthermore, Btk was identified as a downstream target gene of miR-185-5p, suggesting that miR-185-5p may inhibit osteoclast differentiation and migration by targeting Btk. CONCLUSIONS: miR-185 regulates osteoclasts differentiation, with overexpression of miR-185-5p inhibiting osteoclast differentiation and migration in vitro. Additionally, miR-185-5p may modulate osteoclastic differentiation and migration by regulating Btk expression.

Osteoclasts Link Dysregulated Peripheral Degradation Processes and Accelerated Progression in Alzheimer's Disease.

Background: The amyloid-ß (Aß) enhances the number and activity of blood monocyte-derived osteoclasts (OCs). Individuals with osteoporosis (OP) face an increased risk of developing dementia or Alzheimer's disease (AD). Despite this association, the contribution of bone-resorbing OCs to the progression of AD pathology remains unclear. Objective: Our objective was to investigate the potential impacts of OCs on the development of AD pathology. Methods: We conducted targeted analysis of publicly available whole blood transcriptomes from patients with AD to characterize the blood molecular signatures and pathways associated with hyperactive OCs. In addition, we used APP23 transgenic (APP23 TG) AD mouse model to assess the effects of OCs pharmacological blockade on AD pathology and behavior. Results: Patients with AD exhibited increased osteoclastogenesis signature in their blood cells, which appears to be positively correlated with dysfunction of peripheral clearance of Aß mediated by immune cells. Long-term anti-resorptive intervention with Alendronate inhibited OC activity in APP23 mice, leading to improvements in peripheral monocyte Aß-degrading enzyme expression, Aß-deposition, and memory decline. Conclusions: Our findings suggest that OCs have a disease-promoting role in the development and progression of AD, possibly linked to their modulation of peripheral immunity. These findings guide future research to further elucidate the connection between OP and AD pathogenesis, highlighting the potential benefits of preventing OP in alleviating cognitive burden.

Pleurotus sajor-caju (Fr.) Singer ß-1,3-Glucanoligosaccharide (Ps-GOS) Suppresses RANKL-Induced Osteoclast Differentiation and Function in Pre-Osteoclastic RAW 264.7 Cells by Inhibiting the RANK/NFκB/cFOS/NFATc1 Signalling Pathway.

Edible grey oyster mushroom, Pleurotus sajor-caju, ß (1,3), (1,6) glucan possesses a wide range of biological activities, including anti-inflammation, anti-microorganism and antioxidant. However, its biological activity is limited by low water solubility resulting from its high molecular weight. Our previous study demonstrated that enzymatic hydrolysis of grey oyster mushroom ß-glucan using Hevea ß-1,3-glucanase isozymes obtains a lower molecular weight and higher water solubility, Pleurotus sajor-caju glucanoligosaccharide (Ps-GOS). Additionally, Ps-GOS potentially reduces osteoporosis by enhancing osteoblast-bone formation, whereas its effect on osteoclast-bone resorption remains unknown. Therefore, our study investigated the modulatory activities and underlying mechanism of Ps-GOS on Receptor activator of nuclear factor kappa-Β ligand (RANKL) -induced osteoclastogenesis in pre-osteoclastic RAW 264.7 cells. Cell cytotoxicity of Ps-GOS on RAW 264.7 cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and its effect on osteoclast differentiation was determined by tartrate-resistant acid phosphatase (TRAP) staining. Additionally, its effect on osteoclast bone-resorptive ability was detected by pit formation assay. The osteoclastogenic-related factors were assessed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), Western blot and immunofluorescence. The results revealed that Ps-GOS was non-toxic and significantly suppressed the formation of mature osteoclast multinucleated cells and their resorption activity by reducing the number of TRAP-positive cells and pit formation areas in a dose-dependent manner. Additionally, Ps-GOS attenuated the nuclear factor kappa light chain-enhancer of activated B cells' P65 (NFκB-P65) expression and their subsequent master osteoclast modulators, including nuclear factor of activated T cell c1 (NFATc1) and Fos proto-oncogene (cFOS) via the NF-κB pathway. Furthermore, Ps-GOS markedly inhibited RANK expression, which serves as an initial transmitter of many osteoclastogenesis-related cascades and inhibited proteolytic enzymes, including TRAP, matrix metallopeptidase 9 (MMP-9) and cathepsin K (CTK). These findings indicate that Ps-GOS could potentially be beneficial as an effective natural agent for bone metabolic disease.

Circadian Regulation of Bone Remodeling.

Adult bones are continuously remodeled by the balance between bone resorption by osteoclasts and subsequent bone formation by osteoblasts. Many studies have provided molecular evidence that bone remodeling is under the control of circadian rhythms. Circadian fluctuations have been reported in the serum and urine levels of bone turnover markers, such as digested collagen fragments and bone alkaline phosphatase. Additionally, the expressions of over a quarter of all transcripts in bones show circadian rhythmicity, including the genes encoding master transcription factors for osteoblastogenesis and osteoclastogenesis, osteogenic cytokines, and signaling pathway proteins. Serum levels of calcium, phosphate, parathyroid hormone, and calcitonin also display circadian rhythmicity. Finally, osteoblast- and osteoclast-specific knockout mice targeting the core circadian regulator gene Bmal1 show disrupted bone remodeling, although the results have not always been consistent. Despite these studies, however, establishing a direct link between circadian rhythms and bone remodeling in vivo remains a major challenge. It is nearly impossible to repeatedly collect bone materials from human subjects while following circadian changes. In addition, the differences in circadian gene regulation between diurnal humans and nocturnal mice, the main model organism, remain unclear. Filling the knowledge gap in the circadian regulation of bone remodeling could reveal novel regulatory mechanisms underlying many bone disorders including osteoporosis, genetic diseases, and fracture healing. This is also an important question for the basic understanding of how cell differentiation progresses under the influence of cyclically fluctuating environments.

Natural Compounds for Bone Remodeling: A Computational and Experimental Approach Targeting Bone Metabolism-Related Proteins.

Osteoporosis, characterized by reduced bone density and increased fracture risk, affects over 200 million people worldwide, predominantly older adults and postmenopausal women. The disruption of the balance between bone-forming osteoblasts and bone-resorbing osteoclasts underlies osteoporosis pathophysiology. Standard treatment includes lifestyle modifications, calcium and vitamin D supplementation and specific drugs that either inhibit osteoclasts or stimulate osteoblasts. However, these treatments have limitations, including side effects and compliance issues. Natural products have emerged as potential osteoporosis therapeutics, but their mechanisms of action remain poorly understood. In this study, we investigate the efficacy of natural compounds in modulating molecular targets relevant to osteoporosis, focusing on the Mitogen-Activated Protein Kinase (MAPK) pathway and the gut microbiome's influence on bone homeostasis. Using an in silico and in vitro methodology, we have identified quercetin as a promising candidate in modulating MAPK activity, offering a potential therapeutic perspective for osteoporosis treatment.

Sclerostin blockade inhibits bone resorption through PDGF receptor signaling in osteoblast lineage cells.

While sclerostin-neutralizing antibodies (Scl-Abs) transiently stimulate bone formation by activating Wnt signaling in osteoblast lineage cells, they exert sustained inhibition of bone resorption, suggesting an alternate signaling pathway by which Scl-Abs control osteoclast activity. Since sclerostin can activate platelet-derived growth factor receptors (PDGFRs) in osteoblast lineage cells in vitro and PDGFR signaling in these cells induces bone resorption through M-CSF secretion, we hypothesized that the prolonged anticatabolic effect of Scl-Abs could result from PDGFR inhibition. We show here that inhibition of PDGFR signaling in osteoblast lineage cells is sufficient and necessary to mediate prolonged Scl-Ab effects on M-CSF secretion and osteoclast activity in mice. Indeed, sclerostin coactivates PDGFRs independently of Wnt/ß-catenin signaling inhibition, by forming a ternary complex with LRP6 and PDGFRs in preosteoblasts. In turn, Scl-Ab prevents sclerostin-mediated coactivation of PDGFR signaling and consequent M-CSF upregulation in preosteoblast cultures, thereby inhibiting osteoclast activity in preosteoblast/osteoclast coculture assays. These results provide a potential mechanism explaining the dissociation between anabolic and antiresorptive effects of long-term Scl-Ab.

Repurposing the multiple sclerosis drug Siponimod for osteoporosis treatment.

Osteoporosis is the most common bone disorder, in which an imbalance between osteoclastic bone resorption and osteoblastic bone formation disrupts bone homeostasis. Osteoporosis management using anti-osteoclastic agents is a promising strategy; however, this remains an unmet need. Sphingosine-1-phosphate (S1P) and its receptors (S1PRs) are essential for maintaining bone homeostasis. Here, we identified that Siponimod, a Food and Drug Administration-approved S1PR antagonist for the treatment of multiple sclerosis, shows promising therapeutic effects against osteoporosis by inhibiting osteoclast formation and function. We found that Siponimod inhibited osteoclast formation in a dose-dependent manner without causing cytotoxicity. Podosome belt staining and bone resorption assays indicated that Siponimod treatment impaired osteoclast function. Western blot and qPCR assays demonstrated that Siponimod suppressed the expression of osteoclast-specific markers, including C-Fos, Nftac1, and Ctsk. Mechanistically, we validated that Siponimod downregulated receptor activator of nuclear factor kappa B ligand (RANKL)-induced Mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF-κB) signaling pathways during osteoclastogenesis. Moreover, in a preclinical mouse model, Siponimod prevented ovariectomy-induced bone loss by suppressing osteoclast activity in vivo. Collectively, these results suggest that Siponimod could serve as an alternative therapeutic agent for the treatment of osteoporosis.

Chaperone-mediated autophagy protects the bone formation from excessive inflammation through PI3K/AKT/GSK3ß/ß-catenin pathway.

Multiple regulatory mechanisms are in place to ensure the normal processes of bone metabolism, encompassing both bone formation and absorption. This study has identified chaperone-mediated autophagy (CMA) as a critical regulator that safeguards bone formation from the detrimental effects of excessive inflammation. By silencing LAMP2A or HSCA8, we observed a hindrance in the osteoblast differentiation of human bone marrow mesenchymal stem cells (hBMSCs) in vitro. To further elucidate the role of LAMP2A, we generated LAMP2A gene knockdown and overexpression of mouse BMSCs (mBMSCs) using adenovirus. Our results showed that LAMP2A knockdown led to a decrease in osteogenic-specific proteins, while LAMP2A overexpression favored the osteogenesis of mBMSCs. Notably, active-ß-catenin levels were upregulated by LAMP2A overexpression. Furthermore, we found that LAMP2A overexpression effectively protected the osteogenesis of mBMSCs from TNF-α, through the PI3K/AKT/GSK3ß/ß-catenin pathway. Additionally, LAMP2A overexpression significantly inhibited osteoclast hyperactivity induced by TNF-α. Finally, in a murine bone defect model, we demonstrated that controlled release of LAMP2A overexpression adenovirus by alginate sodium capsule efficiently protected bone healing from inflammation, as confirmed by imaging and histological analyses. Collectively, our findings suggest that enhancing CMA has the potential to safeguard bone formation while mitigating hyperactivity in bone absorption.

Conditional loss of CaMKK2 in Osterix-positive osteoprogenitors enhances osteoblast function in a sex-divergent manner.

Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a multi-functional, serine/threonine protein kinase with predominant roles in inflammation, systemic energy metabolism, and bone remodeling. We previously reported that global ablation of CaMKK2 or its systemic pharmacological inhibition led to bone mass accrual in mice by stimulating osteoblasts and inhibiting osteoclasts. However, a direct, cell-intrinsic role for the kinase in the osteoblast lineage has not been established. Here we report that conditional deletion of CaMKK2 from osteoprogenitors, using the Osterix 1 (Osx1) - GFP::Cre (tetracycline-off) mouse line, resulted in increased trabecular bone mass due to an acute stimulation of osteoblast function in male and female mice. The acute simulation of osteoblasts and bone formation following conditional ablation of osteoprogenitor-derived CaMKK2 was sustained only in female mice. Periosteal bone formation at the cortical bone was enhanced only in male conditional knockout mice without altering cortical bone mass or strength. Prolonged deletion of CaMKK2 in early osteoblasts was accompanied by a stimulation of osteoclasts in both sexes, indicating a coupling effect. Notably, alterations in trabecular and cortical bone mass were absent in the doxycycline-removed "Cre-only" Osx1-GFP::Cre mice. Thus, the increase in osteoblast function at the trabecular and cortical bone surfaces following the conditional deletion of CaMKK2 in osteoprogenitors is indicative of a direct but sex-divergent role for the kinase in osteoblasts.

The Species Effect: Differential Sphingosine-1-Phosphate Responses in the Bone in Human Versus Mouse.

The deterioration of osteoblast-led bone formation and the upregulation of osteoclast-regulated bone resorption are the primary causes of bone diseases, including osteoporosis. Numerous circulating factors play a role in bone homeostasis by regulating osteoblast and osteoclast activity, including the sphingolipid-sphingosine-1-phosphate (S1P). However, to date no comprehensive studies have investigated the impact of S1P activity on human and murine osteoblasts and osteoclasts. We observed species-specific responses to S1P in both osteoblasts and osteoclasts, where S1P stimulated human osteoblast mineralisation and reduced human pre-osteoclast differentiation and mineral resorption, thereby favouring bone formation. The opposite was true for murine osteoblasts and osteoclasts, resulting in more mineral resorption and less mineral deposition. Species-specific differences in osteoblast responses to S1P were potentially explained by differential expression of S1P receptor 1. By contrast, human and murine osteoclasts expressed comparable levels of S1P receptors but showed differential expression patterns of the two sphingosine kinase enzymes responsible for S1P production. Ultimately, we reveal that murine models may not accurately represent how human bone cells will respond to S1P, and thus are not a suitable model for exploring S1P physiology or potential therapeutic agents.

Therapeutic avenues in bone repair: Harnessing an anabolic osteopeptide, PEPITEM, to boost bone growth and prevent bone loss.

The existing suite of therapies for bone diseases largely act to prevent further bone loss but fail to stimulate healthy bone formation and repair. We describe an endogenous osteopeptide (PEPITEM) with anabolic osteogenic activity, regulating bone remodeling in health and disease. PEPITEM acts directly on osteoblasts through NCAM-1 signaling to promote their maturation and formation of new bone, leading to enhanced trabecular bone growth and strength. Simultaneously, PEPITEM stimulates an inhibitory paracrine loop: promoting osteoblast release of the decoy receptor osteoprotegerin, which sequesters RANKL, thereby limiting osteoclast activity and bone resorption. In disease models, PEPITEM therapy halts osteoporosis-induced bone loss and arthritis-induced bone damage in mice and stimulates new bone formation in osteoblasts derived from patient samples. Thus, PEPITEM offers an alternative therapeutic option in the management of diseases with excessive bone loss, promoting an endogenous anabolic pathway to induce bone remodeling and redress the imbalance in bone turnover.

Osteomesopyknosis associated with a novel ALOX5 variant that impacts the RANKL pathway.

BACKGROUND: Bone tissue homeostasis relies on the coordinated activity of the bone-forming osteoblasts and bone-resorbing osteoclasts. Osteomesopyknosis is considered a distinctive rare sclerosing skeletal disorder of unelucidated pathophysiology and presumably autosomal dominant transmission. However, the causal genes are unknown. METHODS: We present a case report encompassing clinical assessments, imaging studies, and whole-exome sequencing analysis, complemented by functional in vitro experiments. RESULTS: This new case of osteomesopyknosis was associated with a missense ALOX5 variant predicted to induce protein misfolding and proteasomal degradation. Transfection experiments demonstrated that the variant was associated with reduced protein levels restored by proteasomal inhibition with bortezomib. Likewise, gene expression analysis showed that the mutated gene was associated with a decreased RANKL/OPG ratio, which is a critical driver of osteoclast precursor differentiation. CONCLUSION: Our data indicate impaired bone resorption as the underlying mechanism of this rare osteosclerosis, implicating ALOX5 pathogenic variants as potential etiological factors.

RUFY4 deletion prevents pathological bone loss by blocking endo-lysosomal trafficking of osteoclasts.

Mature osteoclasts degrade bone matrix by exocytosis of active proteases from secretory lysosomes through a ruffled border. However, the molecular mechanisms underlying lysosomal trafficking and secretion in osteoclasts remain largely unknown. Here, we show with GeneChip analysis that RUN and FYVE domain-containing protein 4 (RUFY4) is strongly upregulated during osteoclastogenesis. Mice lacking Rufy4 exhibited a high trabecular bone mass phenotype with abnormalities in osteoclast function in vivo. Furthermore, deleting Rufy4 did not affect osteoclast differentiation, but inhibited bone-resorbing activity due to disruption in the acidic maturation of secondary lysosomes, their trafficking to the membrane, and their secretion of cathepsin K into the extracellular space. Mechanistically, RUFY4 promotes late endosome-lysosome fusion by acting as an adaptor protein between Rab7 on late endosomes and LAMP2 on primary lysosomes. Consequently, Rufy4-deficient mice were highly protected from lipopolysaccharide- and ovariectomy-induced bone loss. Thus, RUFY4 plays as a new regulator in osteoclast activity by mediating endo-lysosomal trafficking and have a potential to be specific target for therapies against bone-loss diseases such as osteoporosis.

Synthesis of Heterocyclic Ring-Fused Bisnoralcohol Derivatives as Novel Small-Molecule Antiosteoporosis Agents.

A series of heterocyclic ring-fused derivatives of bisnoralcohol (BA) were synthesized and evaluated for their inhibitory effects on RANKL-induced osteoclastogenesis. Most of these derivatives possessed potent antiosteoporosis activities in a dose-dependent manner. Among these compounds, 31 (SH442, IC50 = 0.052 µM) exhibited the highest potency, displaying 100% inhibition at 1.0 µM and 82.8% inhibition at an even lower concentration of 0.1 µM, which was much more potent than the lead compound BA (IC50 = 2.325 µM). Cytotoxicity tests suggested that the inhibitory effect of these compounds on RANKL-induced osteoclast differentiation did not result from their cytotoxicity. Mechanistic studies revealed that SH442 inhibited the expression of osteoclastogenesis-related marker genes and proteins, including TRAP, TRAF6, c-Fos, CTSK, and MMP9. Especially, SH442 could significantly attenuate bone loss of ovariectomy mouse in vivo. Therefore, these BA derivatives could be used as promising leads for the development of a new type of antiosteoporosis agent.

Systematic review of the osteogenic effect of rare earth nanomaterials and the underlying mechanisms.

Rare earth nanomaterials (RE NMs), which are based on rare earth elements, have emerged as remarkable biomaterials for use in bone regeneration. The effects of RE NMs on osteogenesis, such as promoting the osteogenic differentiation of mesenchymal stem cells, have been investigated. However, the contributions of the properties of RE NMs to bone regeneration and their interactions with various cell types during osteogenesis have not been reviewed. Here, we review the crucial roles of the physicochemical and biological properties of RE NMs and focus on their osteogenic mechanisms. RE NMs directly promote the proliferation, adhesion, migration, and osteogenic differentiation of mesenchymal stem cells. They also increase collagen secretion and mineralization to accelerate osteogenesis. Furthermore, RE NMs inhibit osteoclast formation and regulate the immune environment by modulating macrophages and promote angiogenesis by inducing hypoxia in endothelial cells. These effects create a microenvironment that is conducive to bone formation. This review will help researchers overcome current limitations to take full advantage of the osteogenic benefits of RE NMs and will suggest a potential approach for further osteogenesis research.

The role of the Piezo1 channel in osteoblasts under cyclic stretching: A study on osteogenic and osteoclast factors.

OBJECTIVES: Orthodontic tooth movement is a mechanobiological reaction induced by appropriate forces, including bone remodeling. The mechanosensitive Piezo channels have been shown to contribute to bone remodeling. However, information about the pathways through which Piezo channels affects osteoblasts remains limited. Thus, we aimed to investigate the influence of Piezo1 on the osteogenic and osteoclast factors in osteoblasts under mechanical load. MATERIALS AND METHODS: Cyclic stretch (CS) experiments on MC3T3-E1 were conducted using a BioDynamic mechanical stretching device. The Piezo1 channel blocker GsMTx4 and the Piezo1 channel agonist Yoda1 were used 12 h before the application of CS. MC3T3-E1 cells were then subjected to 15% CS, and the expression of Piezo1, Piezo2, BMP-2, OCN, Runx2, RANKL, p-p65/p65, and ALP was measured using quantitative real-time polymerase chain reaction, western blot, alkaline phosphatase staining, and immunofluorescence staining. RESULTS: CS of 15% induced the highest expression of Piezo channel and osteoblast factors. Yoda1 significantly increased the CS-upregulated expression of Piezo1 and ALP activity but not Piezo2 and RANKL. GsMTx4 downregulated the CS-upregulated expression of Piezo1, Piezo2, Runx2, OCN, p-65/65, and ALP activity but could not completely reduce CS-upregulated BMP-2. CONCLUSIONS: The appropriate force is more suitable for promoting osteogenic differentiation in MC3T3-E1. The Piezo1 channel participates in osteogenic differentiation of osteoblasts through its influence on the expression of osteogenic factors like BMP-2, Runx2, and OCN and is involved in regulating osteoclasts by influencing phosphorylated p65. These results provide a foundation for further exploration of osteoblast function in orthodontic tooth movement.