Osteoblast Function Research Articles

Rac1-dependent regulation of osteoclast and osteoblast differentiation by developmentally regulated GTP-binding 2

Multiple small GTPases play crucial roles in bone homeostasis by regulating the differentiation and function of bone cells, including osteoclasts and osteoblasts. Here, we investigated whether developmentally regulated GTP-binding protein 2 (Drg2), a subfamily of the GTPase superfamily, could affect bone mass by regulating osteoclast and osteoblast differentiation. Downregulation of Drg2 using siRNA in bone marrow-derived macrophages inhibited osteoclast differentiation and function and Rac1 activation in vitro. Comparatively, Drg2 downregulation in calvarial-derived osteoprogenitor cells enhanced osteoblast differentiation and function in vitro. Rac1 activation was also suppressed by Drg2 downregulation in osteoprogenitor cells. Both osteoclast and osteoblast differentiation regulated by Drg2 downregulation were restored by suppressing Rac1 activity. Drg2-deficient mice showed increased bone mass due to a dramatic reduction in osteoclast numbers without significantly affecting the number of osteoblasts. Furthermore, Drg2 downregulation strongly inhibited RANKL-induced bone loss in vivo. In summary, Drg2 contributes to bone homeostasis by regulating the differentiation and function of osteoclasts and osteoblasts through Rac1 activation. In particular, the effect of Drg2 on osteoclasts is strong enough to regulate bone mass in vivo; therefore, Drg2 has significant potential for use as a therapeutic target in bone loss-related diseases.

Pleiotropic effects of a recessive Col1a2 mutation occurring in a mouse model of severe osteogenesis imperfecta.

In Europe, approximately 85-90% of individuals with Osteogenesis Imperfecta (OI) have dominant pathogenic variants in the Col1a1 or Col1a2 genes whilst for Asian, especially Indian and Chinese cohorts, this ratio is much lower. This leads to decreased or abnormal Collagen type I production. Subsequently, bone formation is strongly reduced, causing bone fragility and liability to fractures throughout life. OI is clinically heterogeneous, with the severity ranging from mild to lethal depending on the gene and the type and location of the OI-causative variant and the subsequent effect on (pro) collagen type I synthesis. However, the specific effects on the phenotype and function of osteoblasts are not fully understood. To investigate this, one of the OI murine models was used, i.e. the oim/oim (OIM) mice, which closest resembling severely deforming OI in humans. We showed that in OIM, the Col1a2 mutation results in a multifactorial inhibition of the osteogenic differentiation and maturation as well as inhibition of osteoclastogenesis. The phenotype of differentiated OIM osteoblasts also differs from that of wild type mature osteoblasts, with upregulated oxidative cell stress and autophagy pathways. The extracellular accumulation of defective type I collagen fibres contributes to activation of the TGF-β signalling pathway and activates the inflammatory pathway. These effects combine to destabilise the balance of bone turnover, increasing bone fragility. Together, these findings identify the complex mechanisms underlying OI bone fragility in the OIM model of severe OI and can potentially enable identification of clinically relevant endpoints to assess the efficacy of innovative pro-osteogenic treatment for patients with OI.

Phospholipase C β4 promotes RANKL-dependent osteoclastogenesis by interacting with MKK3 and p38 MAPK.

Phospholipase C β (PLCβ) is involved in diverse biological processes, including inflammatory responses and neurogenesis; however, its role in bone cell function is largely unknown. Among the PLCβ isoforms (β1-β4), we found that PLCβ4 was the most highly upregulated during osteoclastogenesis. Here we used global knockout and osteoclast lineage-specific PLCβ4 conditional knockout (LysM-PLCβ4-/-) mice as subjects and demonstrated that PLCβ4 is a crucial regulator of receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation. The deletion of PLCβ4, both globally and in the osteoclast lineage, resulted in a significant reduction in osteoclast formation and the downregulation of osteoclast marker genes. Notably, male LysM-PLCβ4-/- mice presented greater bone mass and fewer osteoclasts in vivo than their wild-type littermates, without altered osteoblast function. Mechanistically, we found that PLCβ4 forms a complex with p38 mitogen-activated protein kinase (MAPK) and MAPK kinase 3 (MKK3) in response to RANKL-induced osteoclast differentiation, thereby modulating p38 activation. An immunofluorescence assay further confirmed the colocalization of PLCβ4 with p38 after RANKL exposure. Moreover, p38 activation rescued impaired osteoclast formation and restored the reduction in p38 phosphorylation caused by PLCβ4 deficiency. Thus, our findings reveal that PLCβ4 controls osteoclastogenesis via the RANKL-dependent MKK3-p38 MAPK pathway and that PLCβ4 may be a potential therapeutic candidate for bone diseases such as osteoporosis.

Tideglusib enhances ALP activity and upregulates RANKL expression in Osteoblast-macrophage Co-cultures within a 3D collagen scaffold.

Tideglusib (Tx) is known for its osteogenic potential, yet its effects on the interplay between osteoblasts and M1 macrophages remain underexplored. This in vitro study aimed to isolate and evaluate both the individual and combined roles of M1 macrophages and osteoblasts in macrophage differentiation and osteoblast function, specifically focusing on how these interactions influence protein expression of osteogenesis and osteoclastogenesis in the presence or absence of Tx. Osteoblast and macrophage cells were co-cultured in direct contact for 24 and 48 h, with or without the presence of Tx. ALP activity, the expression of inflammatory-related genes using RT-qPCR, and histological analyses were performed. Co-culturing osteoblasts and M1 macrophages with Tx increased alkaline phosphatase production, indicative of enhanced osteoblast activity. Histological assessments revealed that Tx treatment contributed to the stability and maintenance of cell morphology within the scaffold, suggesting a supportive environment for cell viability and function. Tx significantly reduced the expression of pro-inflammatory markers, such as TNF-α and IL-1β, in the co-culture at both 24 and 48 h Tx also effectively inhibited osteoclastogenic differentiation in macrophages, thereby diminishing their pro-inflammatory phenotype. Tx increased ALP activity and produced a significant up-regulation of RANKL expression, indicating enhanced osteoblast differentiation and osteoclast activation. Tx mitigates macrophage-driven inflammation. Tx may enhance bone regeneration by modulating inflammatory responses and preserving cell integrity.

The Presence of the Endocannabinoid System in an In Vitro Model of Gorham-Stout Disease and Its Possible Role in the Pathogenesis

Gorham-Stout syndrome (GSD), also known as disappearing bone disease, is an extremely rare bone disorder, characterized by a huge bone loss, which is followed by a lack of new matrix deposition and an excessive proliferation of both blood vessels and lymphatics. Unfortunately, the biological causes of GSD are still unknown. Recent studies that have tried to understand the etiopathogenesis of GSD have been principally focused on the vascular and osteoclastogenic aspects, not considering the possibility of a lack of osteoblast function. Nowadays, a diagnosis is still difficult, and is often made by exclusion of the presence of other pathologies, as well as on radiological evidence, and finally confirmed by histological examination. Treatment also remains a critical issue for clinicians today, who mostly try to control the progression of the disease. Over the last two decades, clear evidence has emerged that the endocannabinoid system plays an important role in bone metabolism, leading scientists to hypothesize that it could be involved in physiological and pathological bone processes. In this work, we analyzed the presence of the ES in a primary cell line of human mesenchymal stem cells derived from a GSD patient for the first time, to understand if and how this complex network may play a role in the pathogenesis of the syndrome. Our preliminary results demonstrated that the ES is also present in the pathological tissue. Moreover, the qRT-PCR analysis showed an altered expression of the different ES components (i.e., CNR1, CNR2, TRPV1, and GPR55). We observed an upregulation of CNR1 and TRPV1 expression, while the opposite trend was noticed for CNR2 and GPR55 expression. Thus, these results could lead us to speculate that possible deregulation of the ES may play an important role in the lack of bone regeneration in GSD patients. However, further studies will be necessary to confirm the role of the ES in the progression of GSD and understand whether the natural components of Cannabis Sativa could play a therapeutic role in the treatment of the disease.

Effects of Hyaluronic Acid on Three Different Cell Types of the Periodontium in a Novel Multi-Culture Cell Plate: An Exploratory Study.

Hyaluronic acid (HA) has received considerable attention in the reconstruction of lost periodontal tissues. HA has been proposed to play a role in cell proliferation, differentiation, migration, and cell-matrix as well as cell-cell interactions. Although various studies have been conducted, further research is needed to expand our knowledge based on HA such as its effects on cell proliferation and osteogenic differentiation. The aim of this study is to assess, in single- and multi-culture plate models, the effect of HA on the proliferation, viability, and function of periodontal ligament fibroblasts, osteoblasts, and gingival epithelial cells. A novel multi-culture cell plate was chosen to simulate a cell-cell communication as close as possible to a real clinical condition in an in vitro setting. We found that HA exclusively enhanced epithelial cell proliferation, while intercellular communication stimulated the proliferation and osteogenic potential of the osteoblasts, independently from HA use. The proliferation and function of the periodontal ligament fibroblasts were not changed by HA or the cellular interplay. The use of multi-culture plates could represent a promising method to investigate and compare dental biomaterials in experiments mimicking an in vivo environment.

Glucocorticoids Alter Bone Microvascular Barrier via MAPK/Connexin43 Mechanisms.

Glucocorticoids (GCs) are standard-of-care treatments for inflammatory and immune disorders, and their long-term use increases the risk of osteoporosis. Although GCs decrease bone functionality, their role in bone microvasculature is incompletely understood. Herein, the study investigates the mechanisms of bone microvascular barrier function via osteoblast-endothelial interactions in response to GCs. The animal data shows that prednisolone (Psl) downregulated the osteoblast function and microvessel number and size. To investigate the role of GCs in bone endothelial barrier function further, a bicellular microfluidic in vitro system is developed and utilized, which consists of three-dimensional (3D) perfusable microvascular structures embedded in collagen I/osteoblast matrix. Interestingly, it is demonstrated that GCs significantly inhibit osteogenesis and microvascular barrier function by interfering with endothelial-osteoblast interactions. This effect is triggered by MAPK-induced phosphorylation of connexin43 (Cx43) at Ser282. Collectively, this study sheds light on microvascular function in bone disorders, as osteoporosis, and permits to capture dynamic changes in endothelial-bone interactions under GCs by dissecting the MAPK/Cx43 mechanism and proposing this as a potential target for bone diseases.

Targeting TRPC channels for control of arthritis-induced bone erosion.

Arthritis leads to bone erosion due to an imbalance between osteoclast and osteoblast function. Our prior investigations revealed that the Ca2+-selective ion channel, Orai1, is critical for osteoclast maturation. Here, we show that the small-molecule ELP-004 preferentially inhibits transient receptor potential canonical (TRPC) channels. While ELP-004 minimally affected physiological RANKL-induced osteoclast maturation in murine bone marrow- and spleen-derived myeloid cells (BMSMCs) and human PBMC-derived cells, it potently interfered with osteoclast maturation driven by TNFα or LTB4. The contribution of TRPC channels to osteoclastogenesis was examined using BMSMCs derived from TRPC4-/- or TRPC(1-7)-/- mice, again revealing preferential interference with osteoclastogenesis driven by proinflammatory cytokines. ELP-004 also reduced bone erosion in a mouse model of rheumatoid arthritis. These investigations reveal TRPC channels as critical mediators of inflammatory bone erosion and provide insight into the major target of ELP-004, a drug currently in preclinical testing as a therapeutic for inflammatory arthritis.

The effects of type 1 and type 2 diabetes mellitus on bone health in chronic kidney disease.

Fracture is an under-recognized but common complication of diabetes mellitus, with an incidence approaching twofold in type 2 diabetes mellitus (T2DM) and up to sevenfold in type 1 diabetes mellitus (T1DM) compared with that in the general population. Both T1DM and T2DM induce chronic hyperglycaemia, leading to the accumulation of advanced glycosylation end products that affect osteoblast function, increased collagen crosslinking and a senescence phenotype promoting inflammation. Together with an increased incidence of microvascular disease and an increased risk of vitamin D deficiency, these factors reduce bone quality, thereby increasing bone fragility. In T1DM, reduced anabolic stimuli as well as the presence of autoimmune conditions might also contribute to reduced bone mass and increased fragility. Diabetes mellitus is the most common cause of kidney failure, and fracture risk is exacerbated when chronic kidney disease (CKD)-related mineral and bone disorders are superimposed on diabetic changes. Microvascular pathology, cortical thinning and trabecular deterioration are particularly prominent in patients with T1DM and CKD, who suffer more fragility fractures than do other patients with CKD. This Review explores the pathophysiology of bone fragility in patients with diabetes mellitus and CKD and discusses techniques to predict fracture and pharmacotherapy that might reduce fracture risk.

The microRNA-455 null mouse shows dysregulated bone turnover

Abstract A wide range of specific microRNAs have been shown to have either positive or negative effects on osteoblast differentiation and function with consequent changes in post-natal bone mass; a number of specific targets have been identified. We previously used CrispR-Cas9 to make a miR-455 null mouse, characterising a behavioural phenotype with age. The current study identifies a bone phenotype, starting in younger animals. At three weeks of age, the miR-455 null mice (both male and female) display increased length of both long bones and vertebrae and whilst this difference diminishes across 1 year, it remains significant. Increased bone formation in vivo is mirrored by an increase in osteogenesis from bone marrow-derived stem cells in vitro. This is accompanied by a decrease in osteoclastogenesis and osteoclast function. MicroCT analyses show increased trabecular bone and less porosity/decreased separation in the miR-455 null mouse suggesting a more dense and stronger bone at three weeks of age, these differences normalise by 1 year. Gain-of-function and loss-of-function datasets shows that FGF18 expression is regulated by miR-455, and FGF18 was validated as a direct target of miR-455. The regulation of FGF18 by miR-455 is a likely mediator of its effect on bone.

Pym-18a, a novel pyrimidine derivative ameliorates glucocorticoid induced osteoblast apoptosis and promotes osteogenesis via autophagy and PINK 1/Parkin mediated mitophagy induction.

Glucocorticoid-induced osteoporosis (GIOP) is the most common type of secondary osteoporosis, marked by reduced bone density and impaired osteoblast function. Current treatments have serious side effects, highlighting the need for new drug candidates. Pyrimidine derivatives have been noted for their potential in suppressing osteoclastogenesis, but their effects on osteogenesis and GIOP remain underexplored. Our recent study identified a novel pyrimidine derivative, Pym-18a, which enhances osteoblast functions. In this study, Pym-18a was found to mitigate the detrimental effects of Dexamethasone (Dex) in osteoblast cells and in GIOP in Balb/C mice. Pretreatment with Pym-18a followed by Dex (100 µM) for 24 h restored osteoblast alkaline phosphatase activity and viability. Pym-18a reduced Dex-induced apoptosis and reactive oxygen species (ROS) generation at cellular and mitochondrial levels and preserved mitochondrial membrane potential. Dex impaired autophagy and mitophagy, but Pym-18a pretreatment increased expression of autophagy markers (LC3II) and mitophagy markers (PINK1, Parkin, TOM20) while decreasing P62 expression. The osteogenic effects of Pym-18a were diminished in the presence of 3-MA (an autophagy inhibitor). In silico studies showed mTOR inhibition by Pym-18a, corroborated by its suppression of Dex-induced mTOR activation. In vivo, Pym-18a (10 mg/kg) significantly improved bone microarchitecture, trabecular connectivity, and strength, and corrected P1NP and CTX levels altered by Dex. Pym-18a also promoted autophagy, mitophagy, and suppressed mTOR activation in GIOP mice. Overall, Pym-18a mitigates detrimental effect of Dex by modulating autophagy and PINK/Parkin-mediated mitophagy through mTOR inhibition, suggesting it as a potential novel therapeutic option for GIOP.

LINC01271 promotes fracture healing via regulating miR-19a-3p/PIK3CA axis

ObjectiveOsteoporosis increases the risk of fragility fractures, impacting patients’ lives. This study aimed to investigate whether LINC01271 was involved in the process of fragility fractures and healing, providing a new perspective for its diagnosis and treatment.MethodsThis study included 94 healthy individuals, 82 patients with osteoporosis, and 85 patients with fragility fractures as subjects. RT-qPCR was used to measure the levels of LINC01271, miR-19a-3p, PIK3CA, and osteogenic differentiation markers in osteoblasts and subjects’ serum. Luciferase reporter assays, RIP experiments, and RNA pull-down assays were utilized to verify the target relationships between LINC01271 and miR-19a-3p, as well as between miR-19a-3p and PIK3CA. Cell proliferation and apoptosis were assessed using CCK-8 assays and flow cytometry.ResultsCompared to the healthy control group, the serum levels of LINC01271 were significantly reduced in patients with osteoporosis and fragility fractures. Furthermore, LINC01271 levels increased with time-dependent fracture healing. In vitro studies indicated that LINC01271 boosted osteoblast proliferation, inhibited apoptosis, and augmented osteogenic differences, whereas its inhibition reverses their effects. LINC01271 and PIK3CA were identified as targets of miR-19a-3p, and overexpression of miR-19a-3p could antagonize the effect of LINC01271 in promoting fracture healing.ConclusionThe results indicated that LINC01271 may play a key role in osteoblast function and fracture healing through its interaction with miR-19a-3p and regulation of PIK3CA.

Efficacy of the Bushen Jianpi Huoxue Formula on Beclin-1/Bcl-2-mediated autophagy and apoptosis in osteoblasts.

The Beclin-1/Bcl-2 complex plays a pivotal role in regulating both autophagy and apoptosis in osteoblasts affected by osteoporosis. This study first investigates whether the Bushen Jianpi Huoxue Formula can enhance the cellular function of osteoblasts. Additionally, it initially explores the functional mechanism of Beclin-1/Bcl-2-related apoptosis. Osteoblasts were isolated from the calvaria of three-day-old Sprague-Dawley female rats. The lyophilized power of the Bushen Jianpi Huoxue Formula was prepared from the following ingredients: Fructus Psoraleae, Epimedii Folium, Desertliving Cistanche, Prepared Rehmannia Radix, Radix paeoniae alba, Astragali Radix, Semen Cuscuta, Radix Salviae miltiorrhizae, Angelica sinensis, and Jujube. The primary components were detected by HPLC-MS. Beclin-1 overexpressed osteoblasts were constructed by transfection. Gene expression and protein level were examined by qRT-PCR and immunoblotting assay. Cell viability, apoptosis, and autophagy were assayed with CCK-8, flow cytometer, MDC staining, and Lyso-Tracker staining, respectively. Osteogenic differentiation was assayed by ALP staining, and mineralization by ARS staining. The complex of Beclin-1/Bcl-2 was detected by immunoprecipitation. The results of this study indicated that the Bushen Jianpi Huoxue Formula could enhance both the proliferative activity, differentiation and mineralization of osteoblasts induced by Beclin-1 overexpression. This may be related to its role in activation of the WNT/β-CATENIN by increasing protein expression of WNT1 and β-CATENIN more than 1-fold. The formula effectively inhibited autophagy rate and apoptosis rate of osteoblasts by 50%. Furthermore, the formula was effective in attenuating endoplasmic reticulum stress by decreasing protein expression of AFT4, CHOP, eIF2α, and GRP78 more than 50%, which may play functions by suppressing the PERK signaling pathway. However, Mif treatment significantly weakened the effects of the formula. Bushen Jianpi Huoxue Formula effectively enhanced the osteogenic activity by inhibiting Beclin-1-induced autophagy instead of the binding of Beclin-1 and Bcl-2. This underscores the formula's multifaceted role in promoting bone health and managing cellular stress, and offers novel insights into its therapeutic potential against osteoporosis.

Inhibition of Mitochondrial Fission Reverses Simulated Microgravity-Induced Osteoblast Dysfunction by Enhancing Mechanotransduction and Epigenetic Modification.

Simulated microgravity (SMG) poses substantial challenges to astronaut health, particularly impacting osteoblast function and leading to disuse osteoporosis. This study investigates the adverse effects of SMG on osteoblasts, focusing on changes in mitochondrial dynamics and their consequent effects on cellular energy metabolism and mechanotransduction pathways. We discovered that SMG markedly reduced the expression of osteoblast differentiation markers and promoted mitochondrial fission, as indicated by an increase in punctate mitochondria, a decrease in mitochondrial length, and a reduction in cristae density. These mitochondrial alterations are linked to elevated reactive oxygen species levels, a decrease in ΔΨm, and a metabolic shift from oxidative phosphorylation to glycolysis, resulting in decreased adenosine triphosphate production, which are all indicative of mitochondrial dysfunction. Our results showed that treatment with mitochondrial division inhibitor-1 (mdivi-1), a mitochondrial fission inhibitor, effectively inhibited these SMG-induced effects, thereby maintaining mitochondrial structure and function and promoting osteoblast differentiation. Furthermore, SMG disrupted critical mechanotransduction processes, by affecting paxillin expression, the RhoA-ROCK-Myosin II pathway, and actin dynamics, which subsequently altered nuclear morphology and disrupted Yes-associated protein signaling. Notably, treatment with mdivi-1 prevented these disruptions in mechanotransduction pathways. Moreover, our study showed that SMG-induced chromatin remodeling and histone methylation, which are epigenetic barriers to osteogenic differentiation, can be reversed by targeting mitochondrial fission, further highlighting the significance of mitochondrial dynamics in osteoblast function in an SMG environment. Therefore, targeting mitochondrial fission emerges as a promising therapeutic strategy to alleviate osteoblast dysfunction under SMG conditions, providing novel approaches to maintain bone health during prolonged space missions and safeguard the astronaut well-being.

Transcriptome and metabolome analysis of osteoblasts identifies disrupted purine metabolism and parathyroid hormone associated pathway induced by P. gingivalis infection.

Porphyromonas gingivalis (P. gingivalis), a major pathogenic bacterium of chronic periodontitis and central player in the onset and subsequent progression of periodontitis, can cause alveolar bone resorption. The osteoblast dysfunction induced by P. gingivalis infection is a crucial pathological process causing bone loss. However, the comprehensive responses of osteoblasts, especially metabolism processes involved in osteoblast dysfunction under P. gingivalis invasion are largely unknown. In the present study, to profile the molecules switched in osteoblast dysfunction caused by P. gingivalis infection, the effect of P. gingivalis invasion on osteoblast differentiation was assessed and investigated through transcriptomics and metabolomics approaches. We found that P. gingivalis infection dramatically impaired osteoblast function. P. gingivalis invasion disrupted homeostasis of phosphorus (Pi)/calcium (Ca2+) and induced robust oxidative stress, cell apoptosis and massive activation of inflammatory response in osteoblasts. Notably, the exposure to P. gingivalis induced the inactivation of endocrines pathways, involved in bone formation, which is characterized by downregulated genes and less accumulated metabolites in "Parathyroid hormone synthesis, secretion and action", its downstream "Wnt signaling pathway" and related Pi/Ca2+ transport. Furthermore, we found that disrupted purine metabolism produced less ATP in P. gingivalis-infected osteoblasts and the reduced ATP may directly inhibit phosphorus transport. Collectively, these results provide a new insight into the molecular changes in P. gingivalis-infected osteoblasts in a comprehensive way.

Exploring the Biological Impact of β-TCP Surface Polarization on Osteoblast and Osteoclast Activity.

β-tricalcium phosphate (β-TCP) is a widely utilized resorbable bone graft material, whose surface charge can be modified by electrical polarization. However, the specific effects of such a charge modification on osteoblast and osteoclast functions remain insufficiently studied. In this work, electrically polarized β-TCP with a high surface charge density was synthesized and evaluated in vitro in terms of its physicochemical properties and biological activity. Polarization was performed to achieve a high surface charge density, which was quantified using a thermally stimulated depolarization current. The proliferation and differentiation of MC3T3-E1 osteoblast-like cells were assessed via WST-8 and alkaline phosphatase assays. Tartrate-resistant acid phosphatase (TRAP) activity and a resorption pit assay were used to evaluate the impact of surface charge on RAW264.7 osteoclast-like cell activity. Polarized β-TCP exhibited a surface charge of 1.3 mC cm-2. Electrically polarized surfaces significantly enhanced osteoblast proliferation and differentiation. TRAP activity assays demonstrated effective osteoclast differentiation of RAW264.7 cells, with enhanced activity observed on charged surfaces. Resorption pit assays further revealed improved osteoclast resorption capacity on β-TCP surfaces with a polarized charge. These findings indicate that β-TCP with a highly dense surface charge promotes osteoblast proliferation and differentiation, as well as osteoclast activity and resorption capacity.

Hypoxia Promotes Osteoclast Differentiation by Weakening USP18-Mediated Suppression on the NF-κB Signaling Pathway

Osteoporosis, a prevalent metabolic bone disorder, is characterized by reduced bone density and increased fracture risk. The pathogenesis of osteoporosis is closely associated with an imbalance in bone remodeling, in which the resorption function of osteoclasts exceeds the formation function of osteoblasts. Hypoxia has been implicated in the promotion of osteoclast differentiation and the subsequent development of osteoporosis. The ubiquitin–proteasome system (UPS) and its regulatory enzymes, deubiquitinating enzymes (DUBs), play a significant role in bone homeostasis. In this study, we investigated the contribution and mechanism of Ubiquitin-specific protease 18 (USP18), a DUB, in osteoclast differentiation under hypoxic conditions. BMDMs and RAW264.7 cells were treated with RANKL to induce osteoclastogenesis and were subjected to overexpression or knockdown of USP18 under normoxic or hypoxia conditions. Osteoclast formation was assessed using TRAP staining, and the expression of osteoclast marker genes was determined using qRT-PCR. The activation of the NF-κB signaling pathway was evaluated using immunoblotting. We found that hypoxia significantly enhanced the differentiation of BMDMs and RAW264.7 cells into osteoclasts, accompanied by a notable downregulation of USP18 expression. The overexpression of USP18 inhibited RANKL-induced osteoclast differentiation, while the knockdown of USP18 promoted that process, unveiling the inhibitory effect of USP18 in osteoclastogenesis. Furthermore, the overexpression of USP18 rescued the hypoxia-induced increase in osteoclast differentiation. Mechanistic insights revealed that USP18 inhibits osteoclastogenesis by suppressing the NF-κB signaling pathway, with a potential target on TAK1 or its upstream molecules. This study indicates that hypoxia promotes osteoclast differentiation through the downregulation of USP18, which, in turn, relieves the suppression of the activation of the NF-κB signaling pathway. The USP18 emerges as a potential therapeutic target for osteoporosis treatment, highlighting the importance of the hypoxia–DUB axis in the pathogenesis of the disease.

Genetic Ablation of p16 Mitigates Premature Osteoporosis Induced by PTHrP Nuclear Localization Sequence and C-terminal Deletion through Inhibition of Cellular Senescence.

Premature osteoporosis due to parathyroid hormone-related peptide (PTHrP) dysfunction presents significant bone health challenges. This study explores the role of p16-mediated cellular senescence in this condition using a Pthrp knock-in (KI) mouse model lacking the nuclear localization sequence and C-terminus of PTHrP. We generated p16⁻⁄⁻KI mice and compared them with wild-type, p16⁻⁄⁻, and KI mice. The genetic ablation of p16 in KI mice extended their lifespan, increased body size, and weight. Micro-CT analysis revealed a significant increase in bone volume, while histological and immunohistochemical studies revealed enhanced chondrocyte proliferation and osteoblast function in p16⁻⁄⁻KI mice. In vitro experiments showed enhanced differentiation capacity and reduced senescence of bone marrow mesenchymal stem cells (BM-MSCs) from p16⁻⁄⁻KI mice. Molecular analyses indicated that p16 knockout partially reversed oxidative stress, DNA damage, and cellular senescence observed in KI mice, as evidenced by upregulated antioxidant enzymes, reduced DNA damage markers, and decreased senescence markers. These findings highlight the critical role of p16-mediated cellular senescence in the premature osteoporosis phenotype of KI mice, suggesting that targeting cellular senescence pathways could offer a promising therapeutic strategy for premature osteoporosis and age-related bone loss. This research provides new insights into the interplay between genetic factors, cellular senescence, and bone metabolism in the context of aging and osteoporosis.

Hydroxyurea blunts mitochondrial energy metabolism and osteoblast and osteoclast differentiation exacerbating trabecular bone loss in sickle cell mice

Sickle cell disease (SCD) is a severe hematological disorder characterized by erythrocyte sickling that causes significant morbidity and mortality. Skeletal complications of SCD include a high incidence of bone loss, especially in vertebrae, leading to fragility fractures that contribute to disease burden. Whether hydroxyurea (HU), a front-line therapy for SCD ameliorates bone disease has not been established. To investigate HU action on SCD-related vertebral defects, we used HU-treated “Townes” mice, an SCD animal model and performed high-resolution micro-computed tomography (µCT) imaging to resolve bone volume and micro-architectural structure of cortical and trabecular bone, the two major compartments contributing to bone mass and strength. Our data revealed that cortical bone was significantly diminished in the vertebrae of skeletally mature (representing adults) and immature (representing children) SCD mice, while only mature mice lost trabecular bone mass. Administration of HU ameliorated cortical bone loss in mature SCD mice, but paradoxically promoted trabecular bone decline in both groups. We further investigated the mechanisms of HU action in wild-type C57BL6/J mice. HU caused dose-dependent trabecular bone loss due to diminished osteoclast and osteoblast function, indicative of a low bone turnover state. Mechanistic investigations in vitro revealed that HU impeded osteoblast-progenitor proliferation and early differentiation, and diminished osteoclastogenic cytokine production, blunting osteoclast formation as well as the activity of mature osteoclasts. HU further, suppressed mitochondrial, but not glycolytic energy metabolism in both differentiating osteoblasts and differentiated osteoclasts. Collectively, these findings reveal that despite ameliorating cortical bone loss, HU inhibits trabecular bone formation and resorption, by suppressing mitochondrial energy metabolism and blunting the differentiation and/or activity of osteoblasts and osteoclasts. Together HU drives a low bone turnover state culminating in trabecular bone loss. Further investigation into HU’s impact on bone in SCD patients is warranted for understanding and managing skeletal complications in this population.

Osteoblastic ferroptosis inhibition by small-molecule promoting GPX4 activation for peri-prosthetic osteolysis therapy

Peri-prosthesis osteolysis (PPO) represents the most severe complication of total joint arthroplasty (TJA) surgery and imposes the primary cause of prosthesis failure and subsequent revision surgery. Antiresorptive therapies are usually prescribed to treat PPO, especially for elderly people. Nevertheless, the efficacy of anti-osteoporotic medications remains constrained. Recent therapeutic strategies to promote periprosthetic osseointegration by restoring osteoblast function are considered more effective approaches. However, the precise mechanism underlying the inhibition of osteogenesis triggered by wear particles remains enigmatic. Herein, we demonstrate that wear particles inhibit osteoblast function by inducing ferroptosis to sabotage extracellular mineralization and arouse periprosthetic osteolysis. The suppression of ferroptosis could significantly rescue osteogenesis thus alleviating PPO. Furthermore, Glutathione Peroxidase 4 (GPX4) has been identified as a key target in regulating osteoblastic ferroptosis. By utilizing virtual screening techniques, we have successfully conducted a comprehensive screening of a natural compound known as Urolithin A (UA), which exhibits remarkable inhibition of osteoblastic ferroptosis while simultaneously promoting the process of osteogenesis through its precise targeting mechanism on GPX4. Meanwhile, UA improves the osteolytic conditions significantly in vivo even when the adjunction of titanium (Ti) nanoparticles. This strategy has great potential in treating peri-prosthesis osteolysis and potentially broadens the scope of clinical therapy.Graphical