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.

Vitamin A - discovery, metabolism, receptor signaling and effects on bone mass and fracture susceptibility.

The first evidence of the existence of vitamin A was the observation 1881 that a substance present in small amounts in milk was necessary for normal development and life. It was not until more than 100 years later that it was understood that vitamin A acts as a hormone through nuclear receptors. Unlike classical hormones, vitamin A cannot be synthesized by the body but needs to be supplied by the food as retinyl esters in animal products and ß-carotene in vegetables and fruits. Globally, vitamin A deficiency is a huge health problem, but in the industrialized world excess of vitamin A has been suggested to be a risk factor for secondary osteoporosis and enhanced susceptibility to fractures. Preclinical studies unequivocally have shown that increased amounts of vitamin A cause decreased cortical bone mass and weaker bones due to enhanced periosteal bone resorption. Initial clinical studies demonstrated a negative association between intake of vitamin A, as well as serum levels of vitamin A, and bone mass and fracture susceptibility. In some studies, these observations have been confirmed, but in other studies no such associations have been observed. One meta-analysis found that both low and high serum levels of vitamin A were associated with increased relative risk of hip fractures. Another meta-analysis also found that low levels of serum vitamin A increased the risk for hip fracture but could not find any association with high serum levels of vitamin A and hip fracture. It is apparent that more clinical studies, including large numbers of incident fractures, are needed to determine which levels of vitamin A that are harmful or beneficial for bone mass and fracture. It is the aim of the present review to describe how vitamin A was discovered and how vitamin A is absorbed, metabolized and is acting as a ligand for nuclear receptors. The effects by vitamin A in preclinical studies are summarized and the clinical investigations studying the effect by vitamin A on bone mass and fracture susceptibility are discussed in detail.

Type 2 diabetic mellitus related osteoporosis: focusing on ferroptosis.

With the aging global population, type 2 diabetes mellitus (T2DM) and osteoporosis(OP) are becoming increasingly prevalent. Diabetic osteoporosis (DOP) is a metabolic bone disorder characterized by abnormal bone tissue structure and reduced bone strength in patients with diabetes. Studies have revealed a close association among diabetes, increased fracture risk, and disturbances in iron metabolism. This review explores the concept of ferroptosis, a non-apoptotic cell death process dependent on intracellular iron, focusing on its role in DOP. Iron-dependent lipid peroxidation, particularly impacting pancreatic ß-cells, osteoblasts (OBs) and osteoclasts (OCs), contributes to DOP. The intricate interplay between iron dysregulation, which comprises deficiency and overload, and DOP has been discussed, emphasizing how excessive iron accumulation triggers ferroptosis in DOP. This concise overview highlights the need to understand the complex relationship between T2DM and OP, particularly ferroptosis. This review aimed to elucidate the pathogenesis of ferroptosis in DOP and provide a prospective for future research targeting interventions in the field of ferroptosis.

Exploration of shared gene signatures and molecular mechanisms between type 2 diabetes and osteoporosis.

Type 2 diabetes mellitus (T2D) and osteoporosis (OP) are systemic metabolic diseases and often coexist. The mechanism underlying this interrelationship remains unclear. We downloaded microarray data for T2D and OP from the Gene Expression Omnibus (GEO) database. Using weighted gene co-expression network analysis (WGCNA), we identified co-expression modules linked to both T2D and OP. To further investigate the functional implications of these associated genes, we evaluated enrichment using ClueGO software. Additionally, we performed a biological process analysis of the genes unique in T2D and OP. We constructed a comprehensive miRNA-mRNA network by incorporating target genes and overlapping genes from the shared pool. Through the implementation of WGCNA, we successfully identified four modules that propose a plausible model that elucidates the disease pathway based on the associated and distinct gene profiles of T2D and OP. The miRNA-mRNA network analysis revealed co-expression of PDIA6 and SLC16A1; their expression was upregulated in patients with T2D and islet ß-cell lines. Remarkably, PDIA6 and SLC16A1 were observed to inhibit the proliferation of pancreatic ß cells and promote apoptosis in vitro, while downregulation of PDIA6 and SLC16A1 expression led to enhanced insulin secretion. This is the first study to reveal the significant roles of PDIA6 and SLC16A1 in the pathogenesis of T2D and OP, thereby identifying additional genes that hold potential as indicators or targets for therapy.

Carboxypeptidase M modulates BMSCs osteogenesis-adipogenesis via the MAPK/ERK pathway: An integrated single-cell and bulk transcriptomic study.

The pathogenesis of osteoporosis (OP) is closely associated with the disrupted balance between osteogenesis and adipogenesis in bone marrow-derived mesenchymal stem cells (BMSCs). We analyzed published single-cell RNA sequencing (scRNA-seq) data to dissect the transcriptomic profiles of bone marrow-derived cells in OP, reviewing 56 377 cells across eight scRNA-seq datasets from femoral heads (osteoporosis or osteopenia n = 5, osteoarthritis n = 3). Seventeen genes, including carboxypeptidase M (CPM), were identified as key osteogenesis-adipogenesis regulators through comprehensive gene set enrichment, differential expression, regulon activity, and pseudotime analyses. In vitro, CPM knockdown reduced osteogenesis and promoted adipogenesis in BMSCs, while adenovirus-mediated CPM overexpression had the reverse effects. In vivo, intraosseous injection of CPM-overexpressing BMSCs mitigated bone loss in ovariectomized mice. Integrated scRNA-seq and bulk RNA sequencing analyses provided insight into the MAPK/ERK pathway's role in the CPM-mediated regulation of BMSC osteogenesis and adipogenesis; specifically, CPM overexpression enhanced MAPK/ERK signaling and osteogenesis. In contrast, the ERK1/2 inhibitor binimetinib negated the effects of CPM overexpression. Overall, our findings identify CPM as a pivotal regulator of BMSC differentiation, which provides new clues for the mechanistic study of OP.

CircCOX6A1 suppresses osteogenic differentiation and aggravates osteoporosis via miR-512-3p/DYRK2 axis.

BACKGROUND: Osteoporosis (OP), characterized by compromised bone integrity and increased fracture risk, poses a significant health challenge. Circular RNAs (circRNAs) have emerged as crucial regulators in various pathophysiological processes, prompting investigation into their role in osteoporosis. This study aimed to elucidate the involvement of circCOX6A1 in OP progression and understand its underlying molecular mechanisms. The primary objective was to explore the impact of circCOX6A1 on bone marrow-derived mesenchymal stem cells (BMSCs) and its potential interactions with miR-512-3p and DYRK2. METHODS: GSE161361 microarray analysis was employed to assess circCOX6A1 expression in OP patients. We utilized in vitro and in vivo models, including BMSC cultures, osteogenic differentiation assays, and an OVX-induced mouse model of OP. Molecular techniques such as quantitative RT-PCR, western blotting, and functional assays like alizarin red staining (ARS) were employed to evaluate circCOX6A1 effects on BMSC proliferation, apoptosis, and osteogenic differentiation. The interaction between circCOX6A1, miR-512-3p, and DYRK2 was investigated through dual luciferase reporter assays, RNA immunoprecipitation, and RNA pull-down assays. RESULTS: CircCOX6A1 was found to be upregulated in osteoporosis patients, and its expression inversely correlated with osteogenic differentiation of BMSCs. CircCOX6A1 knockdown enhanced osteogenic differentiation, as evidenced by increased mineralized nodule formation and upregulation of osteogenic markers. In vivo, circCOX6A1 knockdown ameliorated osteoporosis progression in OVX mice. Mechanistically, circCOX6A1 acted as a sponge for miR-512-3p, subsequently regulating DYRK2 expression. CONCLUSION: This study provides compelling evidence for the role of circCOX6A1 in osteoporosis pathogenesis. CircCOX6A1 negatively regulates BMSC osteogenic differentiation through the miR-512-3p/DYRK2 axis, suggesting its potential as a therapeutic target for mitigating OP progression.

MicroRNAs regulate the vicious cycle of vascular calcification-osteoporosis in postmenopausal women.

Menopause is a normal physiological process accompanied by changes in various physiological states. The incidence of vascular calcification (VC) increases each year after menopause and is closely related to osteoporosis (OP). Although many studies have investigated the links between VC and OP, the interaction mechanism of the two under conditions of estrogen loss remains unclear. MicroRNAs (miRNAs), which are involved in epigenetic modification, play a critical role in estrogen-mediated mineralization. In the past several decades, miRNAs have been identified as biomarkers or therapeutic targets in diseases. Thus, we hypothesize that these small molecules can provide new diagnostic and therapeutic approaches. In this review, we summarize the close interactions between VC and OP and the role of miRNAs in their interplay.

Hydrolyzed egg yolk peptide prevented osteoporosis by regulating Wnt/ß-catenin signaling pathway in ovariectomized rats.

Hydrolyzed egg yolk peptide (YPEP) was shown to increase bone mineral density in ovariectomized rats. However, the underlying mechanism of YPEP on osteoporosis has not been explored. Recent studies have shown that Wnt/ß-catenin signaling pathway and gut microbiota may be involved in the regulation of bone metabolism and the progression of osteoporosis. The present study aimed to explore the preventive effect of the YPEP supplementation on osteoporosis in ovariectomized (OVX) rats and to verify whether YPEP can improve osteoporosis by regulating Wnt/ß-catenin signaling pathway and gut microbiota. The experiment included five groups: sham surgery group (SHAM), ovariectomy group (OVX), 17-ß estradiol group (E2: 25 µg /kg/d 17ß-estradiol), OVX with low-dose YPEP group (LYPEP: 10 mg /kg/d YPEP) and OVX with high-dose YPEP group (HYPEP: 40 mg /kg/d YPEP). In this study, all the bone samples used were femurs. Micro-CT analysis revealed improvements in both bone mineral density (BMD) and microstructure by YPEP treatment. The three-point mechanical bending test indicated an enhancement in the biomechanical properties of the YPEP groups. The serum levels of bone alkaline phosphatase (BALP), bone gla protein (BGP), calcium (Ca), and phosphorus (P) were markedly higher in the YPEP groups than in the OVX group. The LYPEP group had markedly lower levels of alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP) and C-terminal telopeptide of type I collagen (CTX-I) than the OVX group. The YPEP groups had significantly higher protein levels of the Wnt3a, ß-catenin, LRP5, RUNX2 and OPG of the Wnt/ß-catenin signaling pathway compared with the OVX group. Compared to the OVX group, the ratio of OPG/RANKL was markedly higher in the LYPEP group. At the genus level, there was a significantly increase in relative abundance of Lachnospiraceae_NK4A136_group and a decrease in Escherichia_Shigella in YPEP groups, compared with the OVX group. However, in the correlation analysis, there was no correlation between these two bacteria and bone metabolism and microstructure indexes. These findings demonstrate that YPEP has the potential to improve osteoporosis, and the mechanism may be associated with its modulating effect on Wnt/ß-catenin signaling pathway.

Role of epigenetic regulatory mechanisms in age-related bone homeostasis imbalance.

Alterations to the human organism that are brought about by aging are comprehensive and detrimental. Of these, an imbalance in bone homeostasis is a major outward manifestation of aging. In older adults, the decreased osteogenic activity of bone marrow mesenchymal stem cells and the inhibition of bone marrow mesenchymal stem cell differentiation lead to decreased bone mass, increased risk of fracture, and impaired bone injury healing. In the past decades, numerous studies have reported the epigenetic alterations that occur during aging, such as decreased core histones, altered DNA methylation patterns, and abnormalities in noncoding RNAs, which ultimately lead to genomic abnormalities and affect the expression of downstream signaling osteoporosis treatment and promoter of fracture healing in older adults. The current review summarizes the impact of epigenetic regulation mechanisms on age-related bone homeostasis imbalance.

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.

Gli1+ Progenitors Mediate Glucocorticoid-Induced Osteoporosis In Vivo.

For a wide range of chronic autoimmune and inflammatory diseases in both adults and children, synthetic glucocorticoids (GCs) are one of the most effective treatments. However, besides other adverse effects, GCs inhibit bone mass at multiple levels, and at different ages, especially in puberty. Although extensive studies have investigated the mechanism of GC-induced osteoporosis, their target cell populations still be obscure. Here, our data show that the osteoblast subpopulation among Gli1+ metaphyseal mesenchymal progenitors (MMPs) is responsive to GCs as indicated by lineage tracing and single-cell RNA sequencing experiments. Furthermore, the proliferation and differentiation of Gli1+ MMPs are both decreased, which may be because GCs impair the oxidative phosphorylation(OXPHOS) and aerobic glycolysis of Gli1+ MMPs. Teriparatide, as one of the potential treatments for GCs in bone mass, is sought to increase bone volume by increasing the proliferation and differentiation of Gli1+ MMPs in vivo. Notably, our data demonstrate teriparatide ameliorates GC-caused bone defects by targeting Gli1+ MMPs. Thus, Gli1+ MMPs will be the potential mesenchymal progenitors in response to diverse pharmaceutical administrations in regulating bone formation.

The potential role of SNHG16/ miRNA-146a/ TRAF6 signaling pathway in the protective effect of zoledronate against colorectal cancer and associated osteoporosis in mouse model.

Bone fracture as a consequence of colorectal cancer (CRC) and associated osteoporosis (OP) is considered a risk factor for increasing the mortality rate among CRC patients. SNHG16/ miRNA-146a/ TRAF6 signaling pathway is a substantial contributor to neoplastic evolution, progression, and metastasis. Here, we investigated the effect of zoledronate (ZOL) on the growth of CRC and associated OP in a mouse model. Thirty Balb/c mice were divided into Naïve, azoxymethane (AOM)/dextran sodium sulfate (DSS), and ZOL groups. Body weight and small nucleolar RNA host gene 16 (SNHG16) expression, microRNA-146a, and TRAF6 in bone, colon, and stool were investigated. Samples of colon and bone were collected and processed for light microscopic, immunohistochemical staining for cytokeratin 20 (CK20), nuclear protein Ki67 (pKi-67), and caudal type homeobox transcription factor 2 (CDx2) in colon and receptor activator of nuclear factor kB (RANK) and osteoprotegerin (OPG) in bone. A computerized tomography (CT) scan of the femur and tibia was studied. ZOL produced a significant decrease in the expression of SNHG16 and TRAF6 and an increase in miRNA-146a in the colon and bone. ZOL administration improved the histopathological changes in the colon, produced a significant decrease in CK20 and Ki-67, and increased CDx2 expressions. In bone, ZOL prevented osteoporotic changes and tumour cell invasion produced a significant decrease in RANK and an increase in OPG expressions, alongside improved bone mineral density in CT scans. ZOL could be a promising preventive therapy against colitis-induced cancer and associated OP via modulation expression of SNHG16, miRNA-146a, and TRAF6.

Osteoporosis: Emerging targets on the classical signaling pathways of bone formation.

Osteoporosis is a multifaceted skeletal disorder characterized by reduced bone mass and structural deterioration, posing a significant public health challenge, particularly in the elderly population. Treatment strategies for osteoporosis primarily focus on inhibiting bone resorption and promoting bone formation. However, the effectiveness and limitations of current therapeutic approaches underscore the need for innovative methods. This review explores emerging molecular targets within crucial signaling pathways, including wingless/integrated (WNT), bone morphogenetic protein (BMP), hedgehog (HH), and Notch signaling pathway, to understand their roles in osteogenesis regulation. The identification of crosstalk targets between these pathways further enhances our comprehension of the intricate bone metabolism cycle. In summary, unraveling the molecular complexity of osteoporosis provides insights into potential therapeutic targets beyond conventional methods, offering a promising avenue for the development of new anabolic drugs.

Ginsenoside Rc alleviates osteoporosis by the TGF-ß/Smad signaling pathway.

Osteoporosis is a common chronic bone disorder in postmenopausal women. Ginsenosides are primary active components in ginseng and the effects of various ginsenoside variants in osteoporosis treatment have been widely revealed. We planned to explore the impact of ginsenoside Rc on bone resorption in an osteoporosis rat model. We used ovariectomized rats to assess the potential impact of ginsenoside Rc on osteoporosis. µ-CT was implemented for analyzing the microstructure of the distal left femur in rats. H&E staining together with Masson staining were applied for bone histomorphometry evaluation. ELISA kits were implemented to detect serum concentrations of TRACP-5b, OCN, CTX, as well as PINP. Ginsenoside Rc treatment lessened the serum levels of TRACP-5b as well as CTX, while increasing serum levels of OCN, and PINP of OVX rats. Moreover, we found that ginsenoside Rc contributed to the synthesis of type I collagen via increasing Col1a1 and Col1a2 levels in femur tissues of ovariectomized rats. Our findings also revealed that ginsenoside Rc activated the TGF-ß/Smad pathway by increasing TGF-ß as well as phosphorylated Smad2/3 protein levels. Ginsenoside Rc alleviates osteoporosis in rats through promoting the TGF-ß/Smad pathway.

Lactobacillus plantarum 45 activates SHP2 through inhibition of oxidative stress to regulate osteoblast and osteoclast differentiation.

BACKGROUND: The purpose of this study is to observe LP45 (Lactobacillus plantarum 45) to investigate the mechanism by which LP45 attenuates oxidative stress-induced damage and regulates the osteoblast-osteoclast balance. MATERIALS AND METHODS: The oxidative stress level and osteoblast- and osteoclast-related proteins were detected by immunofluorescence staining, Western blotting, ROS fluorescent probe and ELISA. Osteoblast cell proliferation capacity was determined by the CCK-8 assay. X-ray observation and HE staining were used to detect the effect of LP45 on osteoporosis. RESULTS: The expression level of SHP2 and Src was significantly increased, and the expression levels of NOX4, P22, P47, IL-1ß, NLRP3, IRF3, RANK, ß-catenin and INF-ß were inhibited in LP45 group and LPS + LP45 group as compared to those in LPS group. Compared with that in LPS group, the concentration of SOD was increased and the concentration of MDA was decreased in LPS + LP45 group. The protein expressions of OPG, RANKL, RUNX3, RANK and ß-catenin in LP45 group and LPS + LP45 group increased. The protein expressions of NF-κB, CREB and AP-1 in LP45 group and LPS + LP45 group decreased significantly. The results were also confirmed by immunofluorescence staining and ROS fluorescent probe. X-ray observation and HE staining showed that LP45 could inhibit the progression of osteoporosis. CONCLUSION: LP45 can exert its antioxidant effect by inhibiting the production of oxidative stress to activate the SHP2 signaling pathway, thus promoting osteoblast differentiation and repressing osteoclast formation to maintain bone homeostasis and improve bone metabolism.

Apigenin and Rutaecarpine reduce the burden of cellular senescence in bone marrow stromal stem cells.

Introduction: Osteoporosis is a systemic age-related disease characterized by reduced bone mass and microstructure deterioration, leading to increased risk of bone fragility fractures. Osteoporosis is a worldwide major health care problem and there is a need for preventive approaches. Methods and results: Apigenin and Rutaecarpine are plant-derived antioxidants identified through functional screen of a natural product library (143 compounds) as enhancers of osteoblastic differentiation of human bone marrow stromal stem cells (hBMSCs). Global gene expression profiling and Western blot analysis revealed activation of several intra-cellular signaling pathways including focal adhesion kinase (FAK) and TGFß. Pharmacological inhibition of FAK using PF-573228 (5 µM) and TGFß using SB505124 (1µM), diminished Apigenin- and Rutaecarpine-induced osteoblast differentiation. In vitro treatment with Apigenin and Rutaecarpine, of primary hBMSCs obtained from elderly female patients enhanced osteoblast differentiation compared with primary hBMSCs obtained from young female donors. Ex-vivo treatment with Apigenin and Rutaecarpine of organotypic embryonic chick-femur culture significantly increased bone volume and cortical thickness compared to control as estimated by µCT-scanning. Discussion: Our data revealed that Apigenin and Rutaecarpine enhance osteoblastic differentiation, bone formation, and reduce the age-related effects of hBMSCs. Therefore, Apigenin and Rutaecarpine cellular treatment represent a potential strategy for maintaining hBMSCs health during aging and osteoporosis.

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Runt-related transcription factor (RUNX1) is a transcription factor closely involved in hematopoiesis. RUNX1 gene mutation plays an essential pathogenic role in the initiation and development of hematological tumors, especially in acute myeloid leukemia. Recent studies have shown that RUNX1 is also involved in the regulation of bone development and the pathological progression of bone-related diseases. RUNX1 promotes the differentiation of mesenchymal stem cells into chondrocytes and osteoblasts and modulates the maturation and extracellular matrix formation of chondrocytes. The expression of RUNX1 in mesenchymal stem cells, chondrocytes, and osteoblasts is of great significance for maintaining normal bone development and the mass and quality of bones. RUNX1 also inhibits the differentiation and bone resorptive activities of osteoclasts, which may be influenced by sexual dimorphism. In addition, RUNX1 deficiency contributes to the pathogenesis of osteoarthritis, delayed fracture healing, and osteoporosis, which was revealed by the RUNX1 conditional knockout modeling in mice. However, the roles of RUNX1 in regulating the hypertrophic differentiation of chondrocytes, the sexual dimorphism of activities of osteoclasts, as well as bone loss in diabetes mellitus, senescence, infection, chronic inflammation, etc, are still not fully understood. This review provides a systematic summary of the research progress concerning RUNX1 in the field of bone biology, offering new ideas for using RUNX1 as a potential target for bone related diseases, especially osteoarthritis, delayed fracture healing, and osteoporosis.

CYLD alleviates NLRP3 inflammasome-mediated pyroptosis in osteoporosis by deubiquitinating WNK1.

BACKGROUND: Osteoporosis (OP) is the result of bone mass reduction and bone structure disorder. Bone marrow mesenchymal stem cells (BMSCs) are the main source of osteogenic precursor cells involved in adult bone remodeling. The involvement of the deubiquitinating enzyme CYLD in OP has recently been discovered. However, the detailed role and mechanism of CYLD remain unknown. METHODS: The OP mouse model was established by performing ovariectomy (OVX) on mice. Hematoxylin and eosin staining, Masson and Immunohistochemical staining were used to assess pathologic changes. Real-time quantitative PCR, Western blot, and immunofluorescence were employed to assess the expression levels of CYLD, WNK1, NLRP3 and osteogenesis-related molecules. The binding relationship between CYLD and WNK1 was validated through a co-immunoprecipitation assay. The osteogenic capacity of BMSCs was determined using Alkaline phosphatase (ALP) and alizarin red staining (ARS). Protein ubiquitination was evaluated by a ubiquitination assay. RESULTS: The levels of both CYLD and WNK1 were decreased in bone tissues and BMSCs of OVX mice. Overexpression of CYLD or WNK1 induced osteogenic differentiation in BMSCs. Additionally, NLRP3 inflammation was activated in OVX mice, but its activation was attenuated upon overexpression of CYLD or WNK1. CYLD was observed to reduce the ubiquitination of WNK1, thereby enhancing its protein stability and leading to the inactivation of NLRP3 inflammation. However, the protective effects of CYLD on osteogenic differentiation and NLRP3 inflammation inactivation were diminished upon silencing of WNK1. CONCLUSION: CYLD mitigates NLRP3 inflammasome-triggered pyroptosis in osteoporosis through its deubiquitination of WNK1.

Young osteocyte-derived extracellular vesicles facilitate osteogenesis by transferring tropomyosin-1.

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) can undergo inadequate osteogenesis or excessive adipogenesis as they age due to changes in the bone microenvironment, ultimately resulting in decreased bone density and elevated risk of fractures in senile osteoporosis. This study aims to investigate the effects of osteocyte senescence on the bone microenvironment and its influence on BMSCs during aging. RESULTS: Primary osteocytes were isolated from 2-month-old and 16-month-old mice to obtain young osteocyte-derived extracellular vesicles (YO-EVs) and senescent osteocyte-derived EVs (SO-EVs), respectively. YO-EVs were found to significantly increase alkaline phosphatase activity, mineralization deposition, and the expression of osteogenesis-related genes in BMSCs, while SO-EVs promoted BMSC adipogenesis. Neither YO-EVs nor SO-EVs exerted an effect on the osteoclastogenesis of primary macrophages/monocytes. Our constructed transgenic mice, designed to trace osteocyte-derived EV distribution, revealed abundant osteocyte-derived EVs embedded in the bone matrix. Moreover, mature osteoclasts were found to release osteocyte-derived EVs from bone slices, playing a pivotal role in regulating the functions of the surrounding culture medium. Following intravenous injection into young and elderly mouse models, YO-EVs demonstrated a significant enhancement of bone mass and biomechanical strength compared to SO-EVs. Immunostaining of bone sections revealed that YO-EV treatment augmented the number of osteoblasts on the bone surface, while SO-EV treatment promoted adipocyte formation in the bone marrow. Proteomics analysis of YO-EVs and SO-EVs showed that tropomyosin-1 (TPM1) was enriched in YO-EVs, which increased the matrix stiffness of BMSCs, consequently promoting osteogenesis. Specifically, the siRNA-mediated depletion of Tpm1 eliminated pro-osteogenic activity of YO-EVs both in vitro and in vivo. CONCLUSIONS: Our findings suggested that YO-EVs played a crucial role in maintaining the balance between bone resorption and formation, and their pro-osteogenic activity declining with aging. Therefore, YO-EVs and the delivered TPM1 hold potential as therapeutic targets for senile osteoporosis.